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ACTIVATION OF ACETATE IN ACETATE—GROwN METHANOSARCINA THERMOPHILA: PURIFICATION AND CHARACTERIZATION OF ACETATE KINASE
by David John Aceti
Thesis submitted to the Faculty of the
Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in
Anaerobic Microbiology
APP ED: I _ a1..... J. G. Ferry, Chairman T. D. Wilkins
*x ‘ ‘ •'\ ’) 1 ~ LN ..D. R. Dean R. H. White
April, 1989 _ Blacksburg, Virginia I 1 ACTIVATION OF ACETATE IN ACETATE-GROWN METHANOSARCINA THERMOPHILA: PURIFICATION AND CHARACTERIZATION OF ACETATE KINASE by David John Aceti
Committee Chairman, J. G. Ferry Department of Anaerobic Microbiology
(ABSTRACT)
Extracts of acetate-grown Methanosarcina thermoghila were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 umoles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 umoles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an az homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 umoles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP,
GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+. Phosphate acetyltransferase was partially k I
I purified to a specific activity of 333 umoles acetyl-COA/min/mg. Optimal activity of phosphate acetyltransferase was between pH 6.7 and 7.2 and between 30°C and 40°C. Rates of acetate kinase and phosphate acetyltransferase activities were approximately 20-fold less in methanol-grown cells compared to cells grown on either acetate or on methanol in the presence of acetate. Western blots of cell extract proteins showed that acetate—grown cells°synthesized higher quantities of the acetate kinase than did cells grown on methanol.
I h I I I I ACKNOWLEDGEMENTS
iv I
I TABLE OF CONTENTS
REBE Abstract...... ii Acknowledgements...... iv List of Tables...... vi List of Figures...... _...... vii I. Introduction...... l II. Literature Review...... 2 III. Materials and Methods...... 22 IV. Results...... 29 V. Summary and Discussion...... 6l VI. Literature Cited...... 67 VII. Vita...... 78
I v I I I I LIST OF TABLES
Häää l. Enzyme activities in cell-free extracts of acetate—grown Q. thermophila...... 30 2. Purification of acetate kinase form acetate—grown Q. thermophila..37 3. Amino acid composition of Q. thermophila acetate kinase...... 44 4. Kinetic properties of acetate kinase from Q. thermophila...... 51 5. Substrate specificity of acetate kinase from Q. thermophila...... 52 6. Divalent cation specificity of Q. thermophila acetate kinase...... SA
vi LIST OF FIGURES
page
1. Proposed carbon and electron flow during acetate conversion to methane in Q. thermophila strain TM—1...... 10 2. Dependence of acetate kinase activity on Q. thermophila cell extract protein...... 32 3. Dependence of phosphate acetyltransferase activity on Q. thermophila cell extract protein...... 33
4. Anion—exchange FPLC of cell extract from acetate—grown Q. thermophila...... 34
5. ATP affinity chromatography of acetate kinase from Q. thermophila...... 38 6. Denaturing polyacrylamide gel electrophoresis of Q. thermophila acetate kinase at each step of purification...... 39 7. Subunit molecular weight determination of Q. thermophila acetate kinase by denaturing slab gel electrophoresis...... 41 8. Molecular weight determination of native Q. thermophila acetate kinase by gel filtration chromatography...... 42 9. Molecular weight determination of native Q. thermophila acetate kinase by non-denaturing gradient gel electrophoresis...... 43 10. Isoelectric point determination of Q. thermophila acetate kinase by isoelectric focusing...... 45 11. Heat stability of Q. thermophila acetate kinase...... 46 12. Influence of pH on activity of acetate kinase from Q. thermophila ...... 48
13. Effect of Mg2+:ATP concentration on activity of acetate kinase from Q. thermophila...... 49 14. Effect of acetate concentration on activity of acetate kinase from Q. thermophila...... 50 15. Effect of Mg2+ concentration on activity of acetate kinase from Q. thermophila...... 53
16. Influence of pH on activity of phosphate acetyltransferase in cell extracts of Q. thermophila...... 55
vii l 17. Influence of temperature on activity of phosphate acetyltransferase in cell extracts of Q. thermcphila...... 56 18. Activity of Q. thermophila phosphate acetyltransferase with CoA or HS·HTP as substrates...... 58 19. Western blot analysis of acetate kinase in extracts of Q. thermophila grown on acetate or methanol...... 60
_ ‘ viii f I. INTRODUCTION
Acetate is the precursor for the majority of methane produced by
methanogenic bacteria in freshwater environments (23, 62) as well as in biomass digestors (89, 9). However, the process of methanogenesis from acetate is not well characterized in comparison to other methanogenic pathways. Thermodynamic considerations have prompted the proposal of an initial step in which acetate is derivitized to an activated form (27). Additional evidence for the existence of an activation step has
been discovered, including studies indicating that acetyl-Coenzyme A is the substrate for the enzyme believed to catalyze the following step in this pathway (100), and the requirement of ATP and Coenzyme A for methane formation from acetate by cell extracts (51). Thus, strong circumstantial evidence exists for an initial
acetate-activation step in the pathway of conversion of acetate to
' methane and CO2. However, only sketchy descriptions exist of enzymes capable of catalyzing this activation in methanogens and none have been
purified. The goals of this investigation were; (1) The identification, purification, and characterization of such an enzyme or enzymes, if present, in Methanosarcina thermophila strain TM—l; and (2) knowledge of the regulation of these enzymes and the relationship of this regulation to methanogenesis from acetate.
! l I
l II. LITERATURE REVIEW
THE METHANE—PRODUCING BACTERIA
The methane-producing bacteria were a relatively obscure and poorly understood form of life prior to the early 1970's. The study of these organisms, also known as methanogenic bacteria or methanogens, was discouraged by their extreme sensitivity to oxygen and slow rate of growth. In the last two decades, however, the improvement of anaerobic techniques, interest in renewable energy resources, and recognition of their unique place in taxonomy has resulted in a rapid accumulation of knowledge about methanogens. Further study can be expected to increase knowledge of the putative common ancestor of the three branches of life, the technology of deriving useful energy from biomass and chemical wastes, and the nature of life in extreme environments.
The Italian physicist Allessandro Volta first noted the formation of methane in freshwater sediments in 1776 (7). The work of Popoff, Hoppe—Seyler, and others in the latter half of the nineteenth century traced this phenomenon to microorganisms (7). Researchers in the first third of the twentieth century were unable to accomplish the difficult task of isolating methanogens, yet they discovered important principles : of methane formation. Barker and others recognized that methanogens _ : could be only the final link in a consortium of microorganisms I to methane (7). most important converting plant material The E conversions carried out directly by methanogens, the production of : F 2 I
II - - 3 methane from acetate and from hydrogen/carbon dioxide, were first proposed during this time (7) and later confirmed in pure culture. A11 attempts to obtain pure cultures were unsuccessful until 1936 and only three species had been isolated by 1956 (8). The development in the 1950's and 1960's of improved anaerobic techniques and particularly the
Hungate roll—tube isolation procedure were of great assistance to the study of methanogens and other anaerobes (39). Ten species were added to the list of pure cultures by 1979 (5). A major milestone in the study of methanogens was their reclassification as archaeobacteria. Originally dispersed among various bacterial taxonomic groups according to morphology, common physiological characteristics prompted H.A. Barker to classify methanogens as a coherent group in 1956 (8). Using 16S rRNA homologies, Woese and colleagues in 1979 confirmed this relationship and further proposed that methanogens were as unrelated to other bacteria as to eucaryotes and so constituted a third major form of life (5). This new classification, the archaeobacteria, included the extreme halophiles and the thermoacidophiles. Existing knowledge of the biochemistry of methanogenesis has been obtained almost entirely within the past 15 years. The conversion of carbon dioxide to methane has received the major share of study due mainly to its early discovery, environmental importance, and its support of a relatively rapid rate of growth. Acetate, the other substrate of importance in the environment, has received relatively little study due to the difficulty of culturing acetate—using methanogens and their slow rate of growth on this substrate. Virtually TTT’————"“—’——————““‘““““—————“‘“""""""""*"*'*"—·—········------¤¤¤¤¤¤¤¤¤¤¤¤¤¤¤¤¤
_ 4
all information regarding acetate conversion was speculation until the early 1980's. The development by Sowers et al. of a simplified and
rapid method for the growth of acetate-utilizing methanogens using a pH auxostat promoted study in this area (92). It is now known that biological methane formation is a nearly ubiquitous phenomenon in anaerobic environments in which the degradation of organic matter is taking place. Methanogens are the
terminal organisms in microbial food chains which accomplish this degradation. Initially, fermentative organisms convert complex
organics to alcohols, volatile fatty acids, carbon dioxide and H2. The higher alcohols and fatty acids are further broken down by proton- reducing acetogens to acetic acid accompanied by the production of H2. Finally, methanogens metabolize the simple compounds formed by the action of the first two groups. The volatility of methane results in
its removal from the system. An anaerobic environment with a redox potential lower than
-330 mV is required for methanogenesis to occur (88). Freshwater and
marine sediments are the source of the majority of biologically- produced methane (110). Methanogens are also found in the rumen, where as much as 200 liters of methane per day may be produced (115), and in the lower intestinal tracts of animals and humans (66, 65). The digestive systems of termites are a significant source of methane (118). A variety of other anaerobic niches, including geothermal springs and deep—sea hydrothermal vents, have yielded methanogenic isolates (98, 42). I Much biologically—produced methane is eventually taken up, after : I I · I 5 diffusion into aerobic zones, by oxygen—requiring methylotrophic organisms (116). The remainder is released to the atmosphere; this quantity has been estimated at 500-800 million tons per year, representing as much as 0.52 of the total annual production of dry organic matter from photosynthesis (116). Methane may be photolyzed in the atmosphere to CO2, CH2O, and CO, thus completing an important part of the environmental carbon cycle (116).' However, not all methane is photolyzed; evidence suggests that biological sources have contributed to a 112 increase in methane, a "greenhouse" gas, in the troposphere over the past ten years (11). In addition to their significance in the environment, methanogens are of interest as a possible source of renewable energy from biological and chemical wastes. Practical efforts to collect and utilize biologically-generated methane have thus far been small-scale; possibly the most significant is the utilization by sewage treatment plants of methane evolving from sewage digestors for the production of power for pumps and the heating of buildings (115). The economic potential of methanogens may increase as fossil fuels are depleted. More than thirty-seven species of methanogens have been identified, comprising fourteen genera, five families, and three orders
(43). The phylogenetic distance between the three orders, Methanobacteriales, Methanococcales, and Methanomicrobiales, is comparable to that between major branches of the eubacteria (31). A non—methanogenic group, the sulfur—independent extreme halophiles, are more closely related to Methanobacteriales than this order is to other methanogens (31). The methanogens, the extreme halophiles, and the 6 extreme thermophile orders Sulfolobus, Thermoproteales, and
Thermoplasma, make up the archaeobacteria (43).
The high degree of genetic variation among methanogens is reflected in phenotypic diversity. All bacterial morphologies are represented; rod, curved rod, coccus, and aggregate forms. Extreme and moderate thermophiles as well as many mesophiles have been isolated. Approximately equal numbers of Gram positive and Gram negative isolates exist. Cell envelopes vary in composition from protein or glycoprotein, heteropolysaccharide, to a variant of murein termed pseudomurein, or certain combinations of these forms. Pseudomurein differs from eubacterial murein in the substitution of Q- acetyltalosaminuronic acid for muramic acid, in the sequences of amino acids in the glycan polymer crosslinking, and possibly in the type of linkage between the alternating sugar moieties (45, A9). Their lack of murein can be used to advantage in the isolation of methanogens, since they are not susceptible to antibiotics such as penicillin and vancomvcin that affect murein biosynthesis. The polar membrane lipids of methanogens and other archaebacteria also differ from those of eubacteria; those of the former are primarily isopranyl glycerol ether derivatives while the latter are ester-linked glycerol derivatives (24, 105). Biosynthetic pathways of methanogens include some which are similiar to those of eubacteria and some which are unique. Glycogen is a major energy reserve of some methanogens, and classical gluconeogenesis has been found to operate (43). Amino acid synthesis seems to occur by usual pathways (43). An incomplete TCA cycle has
u1 7 been reported and appears to operate, in a reverse fashion, primarily for biosynthetic purposes (33). Unique pathways include the assimilation of cell carbon by species which can utilize CO2 for this purpose (54); a portion of the methanogenic pathway is used to reduce one CO2 to a methyl group, while carbon monoxide dehydrogenase reduces another CO2 to CO. The methyl and carbonyl groups are condensed with coenzyme A (CoA) to produce acetyl-CoA, a building block for larger molecules.
Substrates utilizable for methanogenesis are a small number of simple compounds; acetate, carbon dioxide, hydrogen, formate, methanol, methylated amines, and CO. In addition, ethanol, propanol, and butanol were recently found to serve as hydrogen donors for two strains (114). As the precursor for the majority of methane produced in freshwater environments (23, 62) as well as in biomass digestors (89, 9), acetate may be the most important of these substrates overall. Because only small amounts of energy are derived through methanogenesis, growth rates are slow; doubling times on acetate, the least energy—yie1ding substrate with a standard free energy of -36 kJ/mole, are in the range of 12 hours for one of the most efficient acetate—utilizing species (120). The reduction of carbon dioxide to methane with electrons derived from the oxidation of hydrogen or formate (4H2 + CO2 = CHA + 2H2O or 4HCOOH + CO2 = CH4 + 2H2O + 4CO2) is perhaps the best characterized of _ methanogenic pathways (81). In this process, four successive reductions of CO2 occur as it is passed along a chain of three coenzymes unique to methanogens. In the little-understood initial 2 1 8
_ step, CO2 is reduced to the formyl stage and complexed with the coenzyme methanofuran. The formyl group is then transferred to a second coenzyme, tetrahydromethanopterin, and this complex is converted to methenyl—tetrahydromethanopterin. Electrons donated by the coenzyme FA20, a fluorescing 5-deazaflavin found in all known methanogens, are' used to reduce the methenyl—coenzyme complex to methylene-
tetrahydromethanopterin. A reduction to the methyl form is followed by the transfer of the methyl group to 2-mercaptoethanesulfonic acid (coenzyme M or HS-CoM) forming methyl—S—CoM. This compound is the substrate for methyl—CoM methylreductase, a large complex consisting of multiple protein components, FAD, and 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (81). HS-HTP, previously known as component B, has been shown to be the direct electron donor in a reaction in which CHA and the heterodisulfide CoM-S-S-HTP are formed. The reduction of CH3- S-CoM is an exergonic reaction from which ATP is derived in a process which almost certainly involves a proton-motive force (81, 12, 13). Finally, the heterodisulfide CoM-S-S-HTP is reduced to HS—CoM and HS- HTP, a step which involves in an unknown manner the initialization of another cycle of CO2 reduction. Methanogenesis from methanol (4CH3OH = 3CHa + CO2 + 2H2O) is a simpler and more lucrative process since the early reduction steps of the CO2 pathway, some of which are energetically unfavorable, are
avoided. The methyl group of methanol is transferred directly to CoM by two methyltransferases acting in sequence (107). Methyl-S-CoM is
then reduced by methyl-S-CoM methylreductase (36). Three molecules of methanol may be reduced in this manner using reducing potential derived 1 _ 9
from the oxidation of a fourth methanol (A1). Alternatively, electrons may be obtained from another source such as hydrogen (Al). Methanogenesis from methylamines is similiar to that from methanol,
with one methyl group serving as a source of reducing potential for the reduction of three others (lll). Methanogenesis from acetate (CH3COOH = CH4 + 602) will be discussed in the following section.
THE METHANOGENIC DEGRADATION OF ACETATE
Six species, belonging to the genera Methanosarcina and
Methanothrix of the order Methanomicrobiales, have been isolated which are able to use acetate as a methanogenic substrate (A3). Methanogens of other genera may use or even require acetate as a carbon source (21, 3A), but do not significantly convert it to methane. In the proposed pathway (Figure 1), acetate is taken into the cell
by non-active or facilitated diffusion, since the low energy yield of acetate as a substrate would seem to rule out active transport (86).
Further evidence for non-active transport is the demonstration in Methanothrix soehngenii of a cellular KS for acetate similiar to the Km of acetate-CoA ligase, an enzyme which is believed to bind acetate inside the cell (A8). Due to thermodynamic considerations predicting that all but the final step of the pathway are unfavorable, it has been proposed that an initial derivitization of acetate to an activated form occurs (27). Evidence for an activation step is now strong and includes: (1) the 10
P04= CoASH ADP ATP #CH3*C0SCoA <—IE>>———¢¢:i——- #CH3*C0P04= <——:E>———¢ff; #cH3*coo‘ (AK) (PAT) » *co2 (CODH) CoASH (CODH) H20 Fdo Fdf |ääääEä|* (MEMBRANE) > §§FeNi- C0 Eäääääl H2 2H+ ääääää Hscom H2 2H+
CQDH I pcoMPLEx(MT) (MR) (H2ASE)
Figure 1. Proposed carbon and electron flow during acetate conversion to methane in M. thermoghila strain TM-1 (1). AK, acetate kinase: PAT, phosphate acetyltransferase: CODH, carbon monoxide dethydrogenase; Fd, ferredoxin: H2ASE, hydrogenase; MT, methyl transferase, MR, methylreductase. 11
requirement of ATP and CoA for methane formation from acetate by cell
extracts of Methanosarcina barkeri (51); (2) results of electron paramagnetic resonance (EPR) studies indicating that acetyl-CoA is the
substrate of the Methanosarcina thermophila CO dehydrogenase, the enzyme believed to catalyze the following step in the pathway (100); (3) the inhibition of methanogenesis by extracts of Q. barkeri by a compound believed to uncouple at the point of acetate activation (55); and (A) the existence of enzymes capable of activating acetate to acetyl-CoA in acetate-grown cells of Q. barkeri (acetate kinase and
phosphate acetyltransferase;A7) and Q. soehngenii (acetate-CoA ligase;A8).
The activated form of acetate, most probably acetyl·CoA, is bound
by an enzyme complex with CO dehydrogenase activity (103, 100). In Q. thermophila, this five-subunit, 1,000,000 molecular weight complex makes up greater than 102 of the cell protein and contains a corrinoid and a Ni—Fe center (103, 100). Several lines of evidence, including the following, have demonstrated the involvement of the CO dehydrogenase complex: (1) antibodies against CO dehydrogenase
inhibited methanogenesis from acetate by extracts of Q. barkeri (51); (2) methanogenesis from acetate by extracts of Q. barkeri can be
inhibited by propyl iodide, which inhibits corrinoid enzymes (28); (3) CO dehydrogenase activity is 6-fold higher in acetate—grown cells of Q. thermophila in comparison to methanol-grown cells (103). In the central reaction of the pathway, this enzyme complex is proposed to
cleave the carbon-carbon bond of acetate, binding the methyl group to the corrinoid (103) and the carbonyl to the Ni—Fe center (103, 100). 12
This cleavage step may be driven by a Na+ gradient which has been shown to be necessary for methanogenesis from acetate but which has not been assigned a definite function (74). The methyl group is transferred from the corrinoid (possibly via unknown intermediates, such as a proposed second corrinoid; 55) to HS-CoM to form CH3-S—CoM (61).
Electrons derived from the oxidation of the corrinoid-bound carbonyl to carbon dioxide are believed to supply an electron transport chain (101). Little is known about this putative electron transport chain. Recent evidence indicates that in Q. thermophila the electrons are passed directly from the CO dehydrogenase to a ferredoxin (101). A ferredoxin purified from this organism is a trimer of total molecular weight 16,400 containing an uncharacterized Fe-S center (102).
Cytochromes are also possible components of the chain; two b—type and a c-type cytochrome have been identified in membranes of Q. thermophila, and a b—type cytochrome was present in greater amounts in membranes of acetate-grown than methanol-grown Q. barkeri (52). The electron carrier Factor FA20, for which a role in methanogenesis from acetate utilization has not been discovered, may also be a component.
The possible involvement of hydrogenases in this electron transfer has been a subject of speculation. It has been known for some time that both Q. thermophila (60) and Q. barkeri (14) generate a small amount of hydrogen and maintain hydrogen partial pressure within a . certain range (16-92 Pa for Q. thermophila). A H2-cycling system has been hypothesized in which electrons released as H2 by a membrane-bound hydrogenase are recovered by a cytoplasmic hydrogenase (101). A 13 1 1 v hydrogen-evolving system which may reconstruct a part of this proposed scheme has been demonstrated using CO and the following components derived from Q. thermophila; purified CO dehydrogenase, purified ferredoxin, and purified Q. thermophila membranes with an associated hydrogenase (101). Evidence indicating that a b—type cytochrome is linked to the membrane—bound hydrogenase of Q. thermophila (101) further suggests the involvement of both the cytochrome and the hydrogenase. In opposition to the H2-cycling theory, it has been
proposed that the hydrogenase(s) present in acetate-utilizing methanogens may function to rid the cell of excess reducing equivalents or may be involved in anabolic metabolism (60, 14, 101). The latter theory is supported by the demonstration that maintenance of cultures
of Q. barkeri at low H2 partial pressure (thereby forcing the cells to produce H2) does not effect the stoichiometry of acetate degradation (14). Methane evolves from the acetate pathway, as from other
methanogenic pathways, through the reduction of CH3—S—CoM by methyl- CoM methylreductase (61, 69). Although the mechanism of this enzyme is not as well understood in acetate-utilizing as in CO2—utilizing species, a similiar formation of CoM—S-S—HTP and CHA followed by a reduction of the heterodisulfide seems likely. The reducing equivalents used here are necessarily those derived from the oxidation
of CO. Strong evidence indicates that ATP derived from acetate
degradation, as from other methanogenic pathways, is by a chemiosmotic mechanism (15, 55, 74). It has been proposed that the electrochemical _ 14 potential driving the synthesis of ATP may be generated at either of the following steps: (1) the methyl—CoM methylreductase-catalyzed step (13, 55) when coupled to either H2 oxidation or an electron transport chain; or (2) the oxidation of CO to CO2 (15) .
The regulation of the acetate pathway is not well understood. It has been shown that in M. thermophila methanogenesis from acetate is repressed by preferred substrates such as methanol (120). The mechanism of repression is unknown at this time, and could involve competition for a common pathway component, inhibition of acetate- catabolizing enzymes, or repression of acetate-catabolizing enzymes (121). The research described in this thesis focuses on enzymes which may catalyze the initial activation step of the acetate pathway.
Therefore, a general discussion of enzymes which are capable of ( performing this function follows.
ACETATE—ACTIVATING ENZYHES
The "activation" of acetate to the high-energy compounds acetyl- phosphate or acetyl-CoA at the expense of ATP, or the generation of ATP by the reverse process, is central to the metabolism of many organisms. Acetate is activated to the acetyl-CoA form for use as a biosynthetic building block for complex molecules such as lipids, as well as for such energy-yielding pathways as the TCA cycle (80, 19). Acetyl- phosphate has been implicated as an energy source for transport systems rrrrrrrr--——————f———————————————————————————————————————————————-—---——————------
15 1
utilizing periplasmic binding proteins (38). The ATP—generating
reverse reaction links the fermentation of many complex substrates to substrate level phosphorylation.
Two main enzymatic methods of acetate activation are known. Acetate-CoA ligase [Acetate-CoA ligase (AMP—forming): EC 6.2.1.1.) catalyzes the following reaction:
CH3COO” + ATP + CoA = CH3CO—S—CoA + AMP + PPi
Alternatively, the combination of acetate kinase [ATP:acetate
phosphotransferase: EC 2.7.2.1.] and phosphate acetyltransferase
l [acetyl-CoA: orthophosphate acetyltransferase: EC 2.3.1.8.] can activate acetate to acety1—CoA via an acetyl-phosphate intermediate;
acetate kinase CH3COO” + ATP = CH3CO2PO3= + ADP phosphate acetyltransferase CH3CO2PO3= + CoA = CH3CO—S—CoA + Pi
Acetate-CoA ligase has been detected in the organs of higher eucaryotes, in yeasts, and in a variety of bacteria (59, 32, 19, 72). Acetate kinase and phosphate acetyltransferase are widely distributed among procaryotes (2, 46, 75, 18, 44, 68, 83, 109, 20, 22, 47, 56, 73,
82, 96, 97, 79, 108) and acetate kinase has been reported in yeast 1
1 (so). In E. which is capable of producing both acetate-CoA ligase ggli, 1 and acetate kinase/phosphate acetyltransferase, utilization of these 1 two systems apparently depends on changing (19). metabolic needs 1 1 1 1 1
1 Acetate kinase and phosphate acetyltransferase are used for substrate
level phosphorylation and, when acetate is in high concentration, for
the uptake/activation of acetate for growth. An inducible acetate—CoA ligase, which catalyzes a more energy-expensive and higher—affinity
reaction, primarily scavenges acetate present in low concentrations in
- the media. A similiar situation may exist in Azotobacter vinelandii (64), and it is interesting to speculate that many organisms may utilize two methods of acetate activation in this manner.
ACETATE KINASE
Acetate kinase activity was first observed by F. Lipmann in 1944 as the formation of acetyl-phosphate from acetate and ATP by extracts of Lactobacillus delbrueckii (57). Detection in Clostridium butylicum by
Lipmann (96), in Escherichia poli by Kaplan and Lipmann (46), and in Clostridium kluygeri by Stadtman (96), soon followed. Acetate kinase
activity has since been found in a very wide range of procaryotes and has also been reported in a eucaryote, baker's yeast (50). 1 Purifications of this enzyme from E. ggli (4), Propionibacterium shermanii (2), Veillonella alcalascens (16), Acholeplasma laedlawii (44), Clostridium thermoaceticum (83), Bacillus stearothermophilus (68)
and Rhodopseudomonas palustris (108), have been accomplished.
The several functions which have been ascribed to acetate kinase
can vary by organism and by the mode of energy and carbon metabolism in operation at a particular time within a certain organism. The g 17
Peilonella alcalascens (75) and Acholeplasma laidlawii (44) enzymes are believed to function exclusively in the direction of ATP synthesis, as the final step in substrate level phosphorylation. In Anabaena varibilis, acetate kinase appears to be used solely in the opposite direction for the uptake and activation of acetate for cell carbon (73). Acetate kinase operates in either direction in Aerobacter aerogenes (20), activating acetate for biosynthesis in one direction and functioning in the other for substrate level phosphorylation. Many acetate kinases have the capability of using as substrates phosphorylated nucleotides other than adenosine (AA, 109, 18) and may phosphorylate alternative short—chain organic acids, notably propionate (2, 68, 109). The significance of this substrate non—specificity is unknown. Cooperative binding of one or more substrates, most comonly
ATP, is frequent in acetate kinases from various sources (16, 68, 83). The presence of divalent cations is required for activity (109, 68, 18, A4, 75), the functional nucleotide substrate being a Me2+/nucleotide complex (109). Highest activity is achieved with Mg2+ in most cases, although Mn2+ and other divalent cations may be used (109, 68, 18, hh, 75).
The catalytic mechanism of the E. coli acetate kinase has been studied in some detail. The unusual covalent phosphoryl-enzyme intermediate (4, 76), and catalysis of rapid phosphoryl transfer in the absence of cosubstrate (ATP-ADP and acetyl phosphate-acetate) (3, 112), _ have made it a model for general bacterial phosphoryl transfer and a subject of interest to investigators studying enzyme mechanism. Two schools of thought during the 1970's interpreted data as revealing 18
_ either a random ping-pong or a random sequential type of catalysis. The unusual behavior of the enzyme eventually forced the proposal of
hybrids of the two mechanisms, as represented by the models of
Skarstedt and Silverstein (85) and Webb et al. (112). In the former, catalysis is nonrandom sequential in the direction of ATP production but can proceed by either sequential or ping—pong mechanisms in the opposite direction. In the latter model, each mechanism is functional
in either direction but the sequential catalysis is fast relative to the ping-pong catalysis. Nucleotide substrates are believed to bind at a site distinct from the acetate/acetyl-phosphate binding site in the
active site of the E. ggli enzyme (85). A net steric inversion of the phosphate as it is transferred from donor to final acceptor implies
either a single or triple displacement in the acetate kinase reaction. Since investigators have generally accepted the existence of a covalent
phosphoryl-enzyme intermediate, a triple displacement reaction is
postulated (93). This implies the existence of two sites on the enzyme which sequentially bind the phosphate as it is passed to the accepting substrate. One of these sites appears to be a carboxyl group derived
from a glutamyl residue (3, 104). A sulfhydryl group also appears to be necessary in the active site (80). Whether these results are applicable to other acetate kinases is unknown.
PHOSPHATE ACETYLTRANSFERASE
Phosphate acetyltransferase was first assayed, in extracts of _ 19
Clostridium kluygeri, by E. R. Stadtman in 1951 (97). Much of the fundamental description of this enzyme, including the role of CoA, was also the work of Stadtman (97, 95). Purifications have been accomplished from various organisms (79, 108, 77, 25, 113, 84). Phosphate acetyltransferase is known to be cotranscribed with acetate kinase in Q. gg}; (19) and in Salmonella typhimurium (56). As is the case with acetate kinases, phosphate acetyltransferase function appears to depend on the organism or its mode of energy or carbon metabolism at a particular time (20, 19). Catalysis occurs, according to studies of the clostridial enzyme, by a direct displacement mechanism (37). Many phosphate acetyltransferases are stabilized and activated by NH4+, K+, or other cations, while other ions are inhibitory (94, 79, 108). Reducing agents can also stabilize (79) and/or activate (94). Native molecular weights include 63,000- 75,000 for Q. acidurici (79), 38,000-41,000 for Q. kluygeri (79), 160,000-450,000 for Q. gg}; (84), and 55,000 for Q. palustris (108). The reactions catalyzed by phosphate acetyltransferase may be an important regulatory point in some organisms; the activity of the Q. ggli enzyme is regulated by pyruvate and NADH (99), while CoA is a strong substrate inhibitor of the Q. palustris enzyme (108).
HETHANOSARCINA THERMOPHILA STRAIN TM—1
Methanogenesis from acetate has been studied, for the most part, in Q. thermophila and Q. barkeri. These species are similiar in many I I 20 respects and knowledge concerning acetate degradation by one is often considered applicable to the other. However, the use of Q. thermophila in studying the acetate pathway is preferable for several reasons. First, this species has a doubling time on acetate of 12 hours compared to 24-36 hours for Q. barkeri at the optimal growth temperature for each (120, 87). When Q. thermophila is grown at the lower temperatures required by Q. barkeri, growth rates of the former, a moderate thermophile, drop to within range of the latter (120). This observation lends support to the theory that thermophilic conversion of acetate to methane may be more efficient than mesophilic conversion.
Second, Q. thermophila is unable to significantly metabolize H2/CO2
(120) while Q. barkeri utilizes this substrate; the study of the acetate pathway in the former species is therefore free from this interference. Thus, Q. thermophila is the organism of choice for the study of methanogenesis from acetate. Methanosarcina thermophila strain TM—l was isolated from a 55°C anaerobic sludge digestor by Zinder and Mah in 1979 (120). The aggregate morphology of the isolate suggested the genus Methanosarcina, a classification which was confirmed through rRNA homology by Sowers et al. (91). The species name was prompted by an optimal growth temperature at approximately SOOC with an upper limit below 60°C (120). Q. thermophila is a Gram + organism possessing a thin proteinaceous cell wall surrounded by a thick heteropolysaccharide sacculus (121, 91). It has recently been shown that the polysaccharide layer is lost when the organism is grown in media containing marine NaCl I concentrations (90). This discovery is an important step in the 1 I I I
21 development and use of DNA transfer and other molecular genetic techniques in this species. Q. thermophila exhibits growth at a wide range of pH's peaking at 6.0 - 7.0 (120) and is unusual in requiring the vitamin p-aminobenzoic acid (67). Acetate, methanol, and methylated amines serve as substrates. Doubling times are 12 hours on acetate, 7-10 on methanol, and 5 hours in the presence of both acetate and methanol (120). Growth on methanol-acetate mixtures is biphasic, with methanol the predominant methanogenic substrate in the first phase during which acetate may be used as a source of reducing potential and cell carbon (120).
I I
_. 1II I I u III. MATERIALS AND METHODS
Growth of the organism and preparation of cell-free extracts. Q. thermophila strain TM-1 was grown in 10-liter fermentors with acetate (92), methanol (67), or acetate plus methanol as the carbon and energy source(s) as described previously, and harvested in late log phase. Cell extracts (10 — 49 mg protein/ml) were prepared as described previously (69) except the procedure was done aerobically and 2 mM dithiothreitol replaced 2-mercaptoethanol. Extract was stored in liquid nitrogen until use. In the experiment which required removal of any CoA present in the extract, dialysis of extract was performed by a technique described previously (63). A 0.075 ml volume of cell extract (0.8 mg protein) was dialyzed against 30 ml 50 mM (pH 7.0) TES buffer by placing the extract on a 0.45 micron membrane filter (Nucleopore
Corp., Pleasanton CA) floating on the buffer (TES, N—tris(hydroxymethy1)methyl—2-aminoethanesulfonic acid). The extract was dialyzed for one hour at 5°C. Enzym assays. Assays were routinely performed at 37°C. Specific activities are reported as units (pmoles of product formed/min) per mg protein. A Model 552 spectrophotometer (Perkin-Elmer Corp. Norwalk, Conn.) was used. Two methods were used to assay acetate kinase in the forward _ direction, the enzyme-linked assay detecting ADP formation and the hydroxamate assay detecting acetyl phosphate formation. The enzyme- I linked assay couples ADP formation to the oxidation of NADH (63AO = I I 22 I I 23
6.22 mM'l cm'1) through pyruvate kinase and lactic dehydrogenase (2). The assay mixture (0.5 ml) contained (final concentrations): 200 mM
Tris-HC1 buffer, pH 7.3 (Tris, tris[hydroxymethyl]aminomethane); 200 mM potassium acetate; 1.5 mM ATP; 2 mM MgC12; 2 mM phospho(enol)pyruvate;
0.4 mM NADH; 2 mM dithiothreitol; 9 units of pyruvate kinase; and 26 units of lactic dehydrogenase. The combined activities of the linked enzymes in the mixture were routinely assayed to determine that they were not limiting. Assays were initiated by addition of the indicated amounts of protein to the assay mixture. The hydroxamate assay, an adaptation of the method of Lipmann and Tuttle (58) and Rose (80), utilizes the reaction of acetyl phosphate with hydroxylamine to form
acetyl hydroxamate, which forms a colored complex with trivalent iron.
The following components were combined in a volume of 333 ul (final concentrations): 145 mM Tris—HCl, pH 7.4; 200 mM potassium acetate; 10 mM MgC12; 10 mM ATP; and 705 mM hydroxylamine hydrochloride
(neutralized with KOH before addition). The reaction was initiated with the indicated amounts of protein and stopped after 12 min by the addition of 333 ul of 10% trichloroacetic acid followed by the addition of 333 ul of 2.5% FeCl3 in 2.0 N HCl. The mixture was incubated for 5- 30 min to allow color development, and the absorbance at 540 nm was cm”l, measured (csho = 0.58 mM”l experimentally determined). Acetate kinase was assayed in the reverse direction by linking ATP
formation to the reduction of NADP through hexokinase and glucose-6- phosphate dehydrogenase (16). A reaction mixture (0.5 ml) contained
(final concentrations): 100 mM Tris-HC1, pH 7.4; 5 mM ADP; 10 mM MgCl2; 5.5 mM glucose; 1 mM NADP; 2 mM dithiothreitol; 6 units of hexokinase; _ 24 and 3 units of g1ucose—6—phosphate dehydrogenase. Acetate kinase (0.28 ug protein) was added, and the reaction was initiated by the addition of acetyl phosphate (final concentration 20 mM). Reduction of NADP was cm_l). followed at 340 nm (63AO = 6.22 mM°l Phosphate acetyltransferase was routinely assayed at 37°C by X spectrophotometric determination of the formation of the thioester bond cm”l, of acetyl-CoA at 233 nm (72) (6233 = 5.55 mM”l experimentally determined). Reaction cuvettes contained 2.5 ul of cell extract that had been previously diluted 1:30 or partially purified enzyme previously diluted 1:300 in 100 mM Tris-HC1 buffer (pH 7.3). Reactions were initiated by injection of acety1—phosphate (final concentration 2 mM) into 0.5 ml of assay mixture (100 mM Tris—HCl pH 7.3, 0.4 mM CoA, 10 mM ammonium sulfate, 2 mM dithiothreitol, and cell extract or partially purified enzyme). A second type of assay was used additionally in the study of HS-HTP, in which DTNB (5,5‘-dithiobis[2- nitrobenzoic acid]) was employed to react with reduced (non-acetylated) 3 sulfhydryl groups of CoA or HS-HTP (29). This reaction results in the release of a colored nitrobenzenethiol anion that was assayed at 412 nm. In this method, 200 ul aliquots of a 2 ml volume of reaction mix (20 mM potassium phosphate buffer, pH 8.0, with 2 mM acetyl—phosphate, 0.4 mM CoA or 0.4 mM reduced HS-HTP, and cell extract or partially purified phosphate acetyltransferase) were taken at timed intervals.
The enzyme reaction was stopped by boiling for 5 min following acidification with 5 ul of 1 N acetic acid. The sample was then made alkaline with 8 ul of 1 N sodium hydroxide followed by addition of 290 ul 20 mM potassium phosphate buffer (pH 8.0) and 100 ul DTNB/EDTA 1 I 25l
phosphatebuffer).solution (2 mM DTNB and 6 mM EDTA in 20 mM pH 7.0 potassium
HS—HTP (synthesized and provided by K. Noll), where used, was reduced with 10-fold excess sodium borohydride. All procedures were done anaerobically. Myokinase (EC 2.7.4.3) was assayed using a modification of the enzyme-linked acetate kinase assay in which acetate was deleted and 1.5 . mM AMP was added. Purification of acetate kinase. All steps were performed aerobically at 4°C. Activity was assayed in the forward direction using the enzyme-linked assay. Extract (1.5 ml containing 60-70 mg of protein) from acetate-grown cells was injected onto a Mono-Q HR 10/10 anion-exchange column (Pharmacia, Inc. Piscataway, N.J.) equilibrated with buffer A (50 mM TES, pH 6.8, containing 102 (v/v) ethylene glycol, 10 mM MgCl2, and 2 mM dithiothreitol). A linear gradient from 0.0 to
1.0 M KCl was applied at 0.5 ml/min using a Fast Protein Liquid Chromatography (FPLC) system (Pharmacia) equipped with a model GP—250 gradient programer. Active fractions from several Mono-Q separations of cell extract were pooled and loaded onto a 5.0 x 0.9 cm ATP affinity column previously equilibrated with buffer B (10 mM Tris-acetate, pH 7.4, containing 1.2 M KCl, 1 mM MgCl2, and 2 mM dithiothreitol) at a flow rate of 0.5 ml/min. Contaminating proteins and a small amount of activity assayable by the acetate kinase assay were eluted with 15 ml of buffer B, followed by reequilibration with 10 ml of buffer C (buffer B without KCl). The acetate kinase was then eluted at 0.1 ml/min by the application of 1 ml of buffer C containing 15 mM ATP followed by 10 ml of buffer C without ATP. The enzyme was stored in liquid nitrogen. 26 1
Sucrose—gradient ultra—centrifugation. Sucrose-gradient ultra- centrifugation was done in 10 ml polycarbonate centrifuge tubes containing a bottom layer (1.5 ml) of 702 sucrose solution and an upper layer (7.5 ml) of 202 sucrose. Sucrose solutions were made up in 50 mM TES buffer, pH 7.0, with 10 mM MgCl2. One ml of cell extract was loaded onto the 202 sucrose layer and tubes were centrifuged at 151,000 x g for 90 min. Polyacrylamide gel electrophoresis. Polyacrylamide slab gel electrophoresis was performed using the Laemmli buffer system (53) under denaturing (with sodium dodecyl sulfate) or non—denaturing
(without sodium dodecyl sulfate) conditions. Gels were stained for protein with Coomassie blue R-250 (Bio—Rad Laboratories, Richmond, CA). Western blotting. Polyclonal antibodies against Q. thermophila acetate kinase were raised in New Zealand White rabbits. Samples (40 ug protein each) of purified acetate kinase were electrophoresed on a denaturing polyacrylamide gel. Gel bands containing the enzyme were excised and emulsified with Freund's Complete (first injection) or Incomplete (following injections) Adjuvant and were injected subcutaneously. Four weekly injections were followed after an interval of ten weeks by a final injection. Blood taken three days after the supernatantwasfinal injection was allowed to clot and centrifuged. The
stored, after addition of 0.022 sodium azide, at 4°C for several weeks or at -20OC for longer periods of time. For Western blotting, samples of cell extracts or pure enzyme were electrophoresed on 1 adenaturing122 polyacrylamide gel as described earlier. Conditions for the transfer of proteins to nitrocellulose were as described previously 1
1 1 1 1 27(106).Additional protein—binding sites were blocked with casein and { gelatin (0.52 each in 50 mM potassium phosphate buffer, pH 7.4, containing 120 mM NaCl and 0.12 Nonidet P—40 [Sigma Chemical Co., St. Louis, MO]). The protein band corresponding to acetate kinase was
detected using a 1:40,000 dilution of the anti-acetate kinase antiserum and 1251-labelled goat anti-rabbit IgG (0.635 uCi at 7.9uCi/ug (New England Nuclear Laboratories, Boston, MA)). Analytical. The amino acid composition and the N-terminal sequence of acetate kinase were determined at the Commonwealth of
Virginia Protein and Nucleic Acid Sequencing Facility, University of Virginia, Charlottesville, VA. Samples for amino acid analysis were
hydrolyzed in gaggg for 24 hours with constant boiling HC1 (6N). The phenylisothiocyanate—derivatized amino acids were chromatographed using
a Waters 840 LC System (Waters Associates, Milford, MA.) equipped with
a Shandon 25 x 4.6 cm C18 reverse phase 3—micron column, and were eluted in a sodium acetate-acetonitrile gradient. N-terminal amino
acids were sequenced using a model 470 A gas phase peptide sequencer
(Applied Biosystems, Inc., Foster City, CA.) and the phenylthiohydantoin derivatives identified with an on-line Applied
Biosystems liquid chromatograph. Protein was determined by the method of Bradford (17) using protein dye reagent (Bio—Rad Laboratories) and bovine serum albumin (Sigma) as the standard. _ Materials. The ATP affinity column matrix, 8-(6-aminohexylamino)
5'-adenosine triphosphate conjugated to Sepharose 4B, was purchased
from Pharmacia. Myokinase (from porcine muscle), pyruvate kinase and 28 lactic dehydrogenase (both from rabbit muscle), hexokinase and glucose- 6-phosphate dehydrogenase (both from baker's yeast), were purchased from Sigma. All other chemicals were of reagent grade.
{ I I
IV. RESULTS
Enzyme activities in cell-free extracts of acetate-grown Q. thermophila. All enzymes assayed in Q. thermophila extracts in this work showed no differences in activity whether assayed aerobically or anaerobically, and thus were shown to be air-insensitive. All assays were therefore done aerobically except where noted. Extracts of acetate-grown cells of Q. thermophila did not contain acetate-CoA ligase activity, as no dependence on either CoA or myokinase was found in the acetate-CoA ligase assay (Table 1). Only
small amounts of myokinase activity (0.2 f 0.03 U/mg, with standard error after four determinations) were detected in cell extract (10.6 mg . protein/ml) and dialysis of extract to eliminate any CoA present had no effect on the activity. Small amounts of activity were detected in the absence of either acetate or ATP. The former appears to be an ATPase activity while the basis of the latter is unknown. To eliminate the possibility that the major activity is a carboxyl-activated ATPase, propionate and formate were substituted for acetate, giving 1.5 U/mg
and no detectable activity respectively. Thus, the acetate-, ATP-, and magnesium-dependent activities (first four experiments, Table 1) were acetate kinase. After subtraction of the activities determined in the absence of acetate and in the absence of ATP, the mean acetate kinase } activities of the extracts in Table 1 were 2.7 U/mg and 3.1 U/mg. The
meanextracts(acetate-acetate kinase activity in 5 additional acetate-grown I and ATP-minus activities not subtracted) was 4.9 U/mg. I 29 I I I 1 1 30 1
TABLE 1. Enzyme activities in cell-free extracts of acetate—grown Q; thermophila.
Sp. Activityb: Enzymea --—————-—--·—-—-—-——-—--—-— activity Component assayed deleted egtract 1C extract 2d acetate-CoA ligase none 3.05 t 0.61 3.69 t 0.75 and/or acetate kinase acetate kinase myokinase 3.52 i 0.22 3.70 f 0.09
acetate kinase CoA 3.14 i 0.11 3.18 t 0.32 acetate kinase myokinase, CoA NDQ 3.41 t 0.44 ATPase acetate 0.10 i 0.02 0.43 i 0.02
unknown ATP 0.26 i 0.07 0.28 i 0.03
control acetate, ATP ND 0.00 t 0.00 control magnesium 0.09 t 0.04 0.05 t 0.00 aVarious activities were assayed by deletion of individual assay components from the complete acetate-CoA ligase reaction mixture (modified from Oberlies et al.,72). The complete reaction contained: 100 mM Tricine-KOH, pH 8.2 (Tricine, N- tris[hydroxymethyl]methy1glycine), 60 mM potassium acetate, 0.2 mM CoA, 1.5 mM ATP, 2 mM glutathione, 2 mM MgCl2, 2 mM phospho(enol)pyruvate, 0.4 mM NADH, 48 units of pyruvate kinase, 24 units of myokinase, and 5 units of lactic dehydrogenase. The reaction was initiated with extract as indicated and oxidation of NADH was measured at 340 nm. bExpressed as umoles product/min/mg cell extract protein after 2 determinations, with standard error.
CAssays were initiated with 2.5 ul of 12.8 mg protein/ml extract. dAssays were initiated with 2.5 ul of 10.6 mg protein/ml extract.
€Not determined 1 1 1 . 1 1 31
Boiling of extracts for 5 min resulted in the total loss of acetate kinase activity. The quantity of activity measured by the acetate kinase assay was dependent upon the quantity of cell extract protein used to initiate the assay (Figure 2). The diminishing rate of increase observed in this experiment is probably the result of substrate limitation, as the acetate concentration used in this experiment (15 mM) was less than the Km for acetate later determined for the acetate kinase enzyme. Extracts of acetate-grown cells contained phosphate acetyltransferase activity of 49 umoles of acetyl-CoA formed/min/mg
(mean of determinations in 2 extracts). No activity was detected in the absence of either acetyl-phosphate or CoA. Boiling of extracts for 5 min resulted in the total loss of phosphate acetyltransferase activity. The quantity of activity measured by the phosphate acetyltransferase assay was dependent upon the quantity of cell extract protein used in the assay (Figure 3). Cell extract was subjected to anion-exchange FPLC to determine whether certain of the activities could be resolved (Figure 4). Two peaks of acetate kinase activity eluted, in 0.19 - 0.23 M KCl and 0.27
- 0.34 M KCl, containing respectively 93% and 7% of the activity recovered. The total recovery was 26% of the activity loaded. ATPase activity (data not shown) coeluted with the major acetate kinase peak
(total activity 26% relative to acetate kinase activity) and was not detected in the minor acetate kinase peak. Propionate kinase activity coeluted with acetate kinase but was only 3% relative to acetate kinase activity in the major peak compared to 42% in the minor peak. 1 1 1 32
0.8 O TF 0 ‘ QI) E 0.4 ,0 >|__ G Q /
·IL· 0.0 0.0 0.2 0.4 0.6 0.8 I=>R0TEn»1(mg)
Figure 2. Dependence of acetate kinase activity on Q. thermophila cell extract protein. The enzyme-linked assay was used, except that the { potassium acetate concentration was 15 mM. Quantities of cell extract used to initiate the assay were as indicated. { protein I I I 1 33
1.0 O
/\Cf) · _1j-_2 0.6 E3 O O/ EI- 0.4 2 0.2/O
0.0 0 2 4 6 6 10 12 PROTEIN (ug)
Figure 3. Dependence of phosphate acetyltransferase activity on M. thermophila cell extract protein. The assay was as described in 1 Materials and Methods except the concentration of CoA was 0.2 mM. Quantities of cell extract protein used were as indicated. I - 34 I (-—-) (VI) Im] mw (Ü) HSVNIN E.I.VNOIc:IOHcI Q Q Q 'df Q Q <— O CD O CJ O CI
BooN) O! ••···•7> Ad \I OIJCHOl\ YIJCIGJQ)'Ü Y · ä>§°ä.§°£CI‘ä 4-!I[G
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4;Q ‘, L=•O«-1-4::4-!m «- m¤¤°'S§.äää° ‘x äfgääwé ‘I I m m"‘§ä>.ä-S OE <: ä·@~¤==“* I Q ~+<1J¤¤O3rn:>•—-•cm I *5:2.,,;,.2··—-!··-! I gmonconcomoP') P') «—-· N N v- "“ I (V) EISV83;ISNV&Ii'ILI3OV $.2 I EIJ.‘v’I-IcISOI—I:I I-4„CQ)«!-JI!-4ZE .2 $*.41 I
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The phosphate acetyltransferase activity was partially resolved from acetate kinase.
Activities in cel1—frae extracts of methanol-grown calls. Acetate kinase activity in extracts of methanol-grown calls, at 0.23 U/mg (mean of determinations in 2 extracts, acetate- and ATP-minus activities not subtracted), was approximately 20-fold lass in comparison to extracts of acetate-grown calls. Phosphate acetyltransferase activity in extracts of methanol-grown calls, at 3.0
U/mg (determination in 1 extract), was approximately l7—fold less in comparison to extracts of acetate—grown calls. Mixture of extracts of acetate-grown and methanol-grown calls did not result in any loss of total activity of either acetate kinase or phosphate acetyltransferase, indicating that the lower activity in methanol-grown calls was not the result of inhibition.
Localization of acetate kinase and phosphate acetyltransferasa in cell extracts. Sucrose density-gradient ultra-centrifugation of cell extracts of acetate—grown Q. thermophila was used to separate membranes from soluble proteins. Of the applied acetate kinase (27 Units, enzyme-linked assay) and phosphate acetyltransferasa (306 Units) activities, approximately 772 of the former and 852 of the latter were recovered in the soluble fractions, and no significant activity was detected in the membrane fractions. These results suggest that acetate kinase and phosphate acetyltransferase are not integral membrane proteins; however, a loose association cannot be ruled out. M 36 M M
Purification of acetate kinase. Dithiothreitol (2 mM) was included in buffers at all steps of purification since it was found that incubation of partially purified (by anion—exchange FPLC) preparations of acetate kinase with 2 mM dithiothreitol for 15 min at room temperature resulted in activity approximately 10-fold greater than unincubated preparations and 30 — 40 fold greater than preparations incubated for 15 min with no added dithiothreitol. In addition, a number of investigators (4, 18, 80, 82, 83, 85) have found reducing agents such as dithiothreitol, cysteine, 2—mercaptoethanol, or glutathione useful in stabilizing acetate kinase activity. Table 2 shows a representative purification of the acetate kinase from extracts of acetate-grown Q. thermophila using Mono-Q anion- exchange and ATP affinity chromatography. Representative column profiles are shown in Figures 4 and 5. Only fractions from the Mono-Q step contained within the major acetate kinase peak and with high specific activity were used for further purification by affinity chromatography. When the anion—exchange- and ATP affinity-purified preparation was subjected to denaturing polyacrylamide gel electrophoresis (Figure 6), only one band was observed. Native 7% polyacrylamide gel electrophoresis of the preparation resulted in two distinct bands of slightly different mobilities (data not shown); the trailing band contained an estimated (by staining density) 4-5 fold greater quantity of protein than the faster-migrating band. Acetate kinase activity was detectable in a gel slice corresponding to the M bands which was cut from a native polyacrylamide gel. ATPase activity, M M M M 37
1 >- 1 1 1 1.1 1 I I ¤.1 IG 1-1 ~O1 I >AI® ("7 1--II 1 ON11-1 I I U~.«1 1 1 Q1 1 I 1 cc 1 1 1 1 1 1 1 1 1 Q 1 1 • I Ö I I ZI I -1-1 1 I 11·‘¤m-1.1 1 1 • Q I1-1 1-1 NI 3 11-10 1 1*1 GI A 0 10-1-1 1 1-11 >-, $1 1L=.1I1-1 1 1 m C10 1 -1-I 1 I In 1 1 $-1 1 I In Q} 1 :1 I 1 m -1.1 I Q., 1 1 m I 1 I *¤ 1.1 1 A1 · 1 GJ 01 I<.1:>~.o¤1 1 At O 1--14.1E1 0.50 E 01 Ü 1O TOO O ,11*;Ä] cg ¤ E1 \ }_E E O 0.25 “ E EJ 0 Z:ä E 1, Ää —§:: 0.00 0 0 :::2"4 8 $:2::12 242 : : 2 282:5. 32 ELUENT(mI) Figure 5. ATP affinity chromatography of acetate kinase from M. Combined active fractions from three anion—exchange thermophila.fractionations of cell extract (similar to Fig. A), containing a total of 1.87 mg protein and 22A units (pmoles ADP formed/min) of acetate kinase activity, were chromatographed as detailed in Materials and Methods. The enzyme was eluted by the application of 1 ml of buffer containing 15 mM ATP (arrow). The total activity is reported for each 1 ml fraction. No activity or protein was detected in the deleted portion of the profile.- 1 . 40 Z J which was 142 relative to acetate kinase activity in cell extract and 262 in an anion—exchange partially purified preparation, was 1-22 in the final preparation. No phosphate acetyltransferase activity was Z detectable in the preparation. Properties of purified acetate kinase. A relative mobility (Mr) corresponding to a molecular weight of 53,000 was estimated by denaturing gel electrophoresis (Figure 7). The Mr of the native enzyme corresponded to molecular weights of 94,000 and 87,000 when estimated by gel filtration chromatography (Figure 8) and by native gradient gel electrophoresis (Figure 9), respectively_ In an N-terminal sequence analysis, only one N—terminus was detected: Met-Lys-Val-Leu-Val-Ile- Asn-Ala-Gly—Ser-Ser-unknown-Leu-Lys-Tyr-Gln-Leu. The amino acid composition is shown in Table 3. Two bands corresponding to isoelectric points of 4.5 and 4.7 were observed when the enzyme was subjected to isoelectric focusing (Figure 10). The activity was stable for at least four months when frozen in liquid nitrogen. Activity was stable to heating for 15 min at temperatures of up to 70°C, but was inactivated at higher temperatures (Figure 11). Activity in the reverse direction was 2.6·fold greater than in the forward (hydroxamate assay) direction when the assays were performed as described in Materials and Methods. The latter is presumed to be the physiologically important direction and was studied further. Optimum acetate kinase activity (hydroxamate assay) was between pH 7.0 41 1 In CD 2 0.6 L1.! g2 0.4 L1.! CE Figure 7. Subunit molecular weight determination of M. thermophila acetate kinase by denaturing slab gel electrophoresis. Electrophoresis and detection by Coomassie staining of purified acetate kinase and protein standards on adjacent lanes of a 122 denaturing polyacrylamide gel were as described in Materials and Methods. Symbols: standards from Bio-Rad (lysozyme, 14,400; soybean trypsin inhibitor, 21,500; carbonic anhydrase, 31,000; ovalbumin, 45,000; bovine serum albumin, 66,200; phosphorylase B, 92,500); ·,acetate kinase. , 1 420.8 . 1 1 > O ¥.< 004 O 0.2 · 4.2 4.6 5.0 5.4 LOG MOLECULAR WEIGHT Figure 8. Molecular weight determination of native M. thermophila acetate kinase by gel filtration chromatography. Gel filtration was performed at 0.02 ml/min on a 0.9 x 34.5 cm column of Sephadex G—200 SF (Sigma) equilibrated with 100 mM Tris-HC1 buffer, pH 7.2, containing 200 mM KCl, 1 mM ATP, and 0.1 mM phenylmethylsulfonylflouride (Sigma). The elution of acetateMolecularkinase (·)weightwasstandardsdetected by(())thefromenzyme-linkedPharmacia were activitycatalase assay.(232,000), aldolase (158,000); bovine serum albumin (67,000); ovalbumin (43,000); and chymotrypsinogen A, (25,000). KAV = (Ve — vo)/(vt — VO) where VO equals void volume, Vt equals total volume, and Ve equals elution volume. I 43 I I O 9. Molecular weight determination of native M. thermoghila acetate8Figurekinase by non-denaturing gradient gel electrophoresis. Purified acetate kinase (Q) and molecular weight standards (O) were _ electrophoresed on a non-denaturing 4- 302 polyacrylamide linear gradient gel and detected by Coomassie staining. Molecular weight standards (Behring Diagnostics, San Diego, CA) were cytochrome C (12,500), chymotrypsinogen A (25,000), egg albumin (45,000), black albumin (68,000), hexokinase (100,000), aldolase (158,000), B- glucuronidase (290,000), and ferritin (450,000). 44 Table 3. Amino acid composition of M. thermophila acetate kinase. Amino acida ’ Residues (molZ) Ala 6.4 Arg 4.5 Asp + Asn 8.5 Cys 1.2 Glu + Gln 10.0 Gly 14.7 His 2.4 Ile 6.7 Leu 6.7 Lys 5.5 Met 3.1 Pro 4.1 Phe 3.6 Ser 8.1b um 5. gb Tyr 2.2 Val 6.6 aTryptophan was not determined. bValues were corrected for degradation during acid hydrolysis by extrapolation from standards. 46 1 100<>77—————Q‘~ E 80 \\ 2F- ZD E >< IE ÖQ , 20 O\ O 2 50 40 50 60 70 60 TEMPERATURE (°C) — Figure 11. ‘Heat stability of E. thermophila acetate kinase. Each sample (0.23 ug acetate kinase in 5ul) was added to 10 ul of prewarmed 100 mM Tris—HCl (pH 7.4 at the indicated temperature) and incubated for 15 min. The enzyme solution was immediately cooled to 4°C and the activity determined in the forward direction at 37°C by the enzyme- linked assay, which was initiated with 0.23 ug of enzyme. Activity was 1 88 U/mg protein (1002) after preincubation at 30°C. 1 1 1 1 47 = l1 and 7.4, with little activity below pH 5 or above pH 9 (Figure 12). Hyperbolic saturation curves were obtained with both Mg2+:ATP (Figure 13) and acetate (Figure 14) and no substrate inhibition was observed with up to 10 mM Mg2+:ATP or 250 mM acetate. The kinetic constants for acetate and Mg2+:ATP, shown in Table 4, were determined from double reciprocal plots of substrate concentration versus activity (inset, figures 13 and 14). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate (Table 5). Other tri- phosphorylated nucleotides replaced ATP (Table 5). The enzyme did not show a preference for purine nucleotides. All acetate kinases studied require divalent cations for activity, with Mg2+ providing maximum activity in most cases (109, 68, 18, 44, 75). The Q. thermophila enzyme also required divalent cations and utilized Mg2+ for activity in either direction. Variation of the MgCl2 concentration with a constant concentration of 10 mM ATP resulted in a broad peak of activity with an optimum at 10 mM MgCl2, indicating an optimum Mg2+/ATP of 1.0 (Figure 15). Substitution of Mn2+ for Mg2+ yielded a 302 increase in the rate of activity; substitution with Co2+, Ca2+, Cu2+, Ni2+, and Zn2+ resulted in lower rates or no activity (Table 6). Phosphate acetyltransferase; characterization of activity in cell extracts and partial purification. Phosphate acetyltransferase E ” was optimal between 6.7 and activity in extracts of acetate-grown cells i 7.2 (Figure 16). When activity in extracts was assayed over a broad : temperatures, a peak occurred between 30°C and 40°C range of reaction E (Figure 17) (this enzyme has been shown, in an experiment using : ‘ 48 I I I I ' 1 /°\CJ) 1 E 1 ~/Ü\D I <( 200 C) L1. 04C.) 100 C1. Cf) Figure 12. Influence of pH on activity of acetate kinase from Q. thermophila. The hydroxamate assay was used with the following modifications: Tris buffer was excluded; the potassium acetate concentration was 600 mM; hydroxylamine-HC1 was adjusted to the indicated pH with KOH before addition of the remaining assay components. The reaction was initiated with 0.45 ug of enzyme and was stopped after 5 min. The pH of the assay mixture did not change during the reaction. 49 600 ' G 600 E 3 400 T 0.010 § 0.008 E 300 7 E 0.006 <( 4; 0.00+ U 2ÜO ä ß ä 0.002 0. V7 100 -0.4 0.0 0.4 0.8 1.2 1.6 2.0 [¤02+=^TP]" <¤«¤>" 0 0 2 4 6 8 10 , [Mg2+:ATP] (mM) Figure 13. Effect of Mg2+:ATP concentration on activity of acetate kinase from M. thermophila. The specific activity of 0.12 ug of acetate kinase was measured (hydroxamate assay) with Mg2+:ATP 0.67, 0.80, 1.0, 1.25, 1.4, 2.0, concentrations at 0, 0.25, 0.33, 0.50, ‘A 2.5, 3.33, 4.0, 5.0, 6.25, 8.0, and 10.0 mM. double reciprocal plot (inset) of points at Mg2+:ATP concentrations of 0.5 - 8.0 mM yielded a line with L = 0.998. * 50 1 700 Q; 600 EE 500 „ §° 0.006 400 ,?’\ 2 0.004 *64 ÖOO 0.006 ä é 0.002 Ö 200 ä 0 E 35 U) 100 -6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 102 x [ACETATE]'1 („„¤)—1 0 0 50 100 150 200 250 [ACETATE] (mM) Figure 14. Effect of acetate concentration on activity of acetate kinase from M. thermophila. The specific activity of 0.12 ug of acetate kinase was measured (enzyme—1inked assay) with acetate concentrations at 0, 1 , 2, 3, 4, 7, 10, 15, 20, 25, 30, A0, 50, 70, 100, 130, 160, 200, and 250 mM, with potassium held constant at 250 mM. A double reciprocal plot (inset) of points at acetate concentrations from 10 — 100 mM yielded a line with E = 0.995. I 1 1 ” 1 1 1 I 51 1I I I I I I I I I I I I UNI I I I I In¤.)I I Iß-JS!/II IQ) INI-INI II-I Iä:-I-III.¤U:I II-IIN • I 'U,£I IQ) N Ic:O I IO I—I I I-II1)II·-I¢)I N ~«-I ILIQIILIINIAI J: IID NINQIII-I I>LI-I4.II•—I«-IIO O IOOGII I E I: UI ILI GJLI IDG)ILII/INII IEIID ,.:2 IE-I«—Iq)I I·«-I LJ I OI-—-II I": • I EOI I I \4EI IQ) ZI I I I,.: I I I«I-J E I I I 0 I I ILI LI I I IÜ II-I I : I II-I-I I ·I-I I I QJ I E/·~I I4-J In I ESI I,.: CU I 4-I•I···4I IOC C2 I UIDI I-I-I -I-·I I :III.II IQ) Ad I ><'¤cII I3 I RIO}-IIT\®I cu I EI-I¤.•Ir\©ILI I-I I>¤I |I\&D| N I c0I II-I I:N I IIIEI ID cu I ID I IU U I «-ILII Ir—IIQJ N I OIIJI I EQII IO ¥I—I I 1 I IE O I ~.« I I I I IQ UI I I IC ·I··II1) I.I rx I|®©IC)••I •~ IJ IX INNIQ I-I I xa I ('\lIO\ Q) I I I Q, I I IC O I I IÖ LI I I I O., I I I'¤ I I IIJJ U I I q)IIn ~I-I I I LJIN II-I I ID I NI,-Q cu I II-I I 4-II Cl I N I UI: ·I-I I LI I UIO x I 4.1 I NI·I-I I In I I«I-I I ,¤ I äncu • I Ö IÜA I!*I Q I U) IE•I·II D • I Iäml UO) Q) I••U’JII—IUI ·—I I I+ NI NN „¤ I Ic~I«I-IILJC N I IODOI ·I-I EI I IZI3-IIN.=¢ 52 Table 5. Substrate specificity of acetate kinase from M. thermophila. Substrates Z Activity ATP, acetatea 100 TTP, acetatea 106 ITP, acetatea 83 UTP, acetatea 80 GTP, acetatea 79 CTP, acetatea 53 ATP, acetateb 100 ATP, propionateb 60 ATP, formateb 0 aThe hydroxamate assay was used as described in the text except where the sodium salts of the indicated nucleotides (10 mM) replaced ATP. The reactions were initiated with 0.12 ug of enzyme. Activity with ATP (100Z) was 412 umoles of acetyl phosphate/min per mg protein. bThe enzyme—linked assay was used with the potassium salts of the acids at 200 mM. The reactions were initiated with 0.12 ug of enzyme. Activity with acetate (100Z) was 569 umoles of ADP produced/min per mg protein. 53 1 I 100 — 80 2 E4 60 3 I h E>< 40 I E< 20 I X I 0 0.0 1.0 2.0 3.0 M 2+/ATP RATI0 Figure 15. Effect of the Mg2+ concentration on activity of acetate kinase from Q. thermophila. The hydroxamate assay was used with a constant ATP concentration of 10 mM and an amount of MgC12 to provide the indicated ratio of Mg2+:ATP. The reaction was initiated with 0.12 ug of enzyme. Maximal (1002) activity was 598 umoles of U acetyl phosphate/min/mg protein. I I I I I I 11 1 _ 54 1 Table 6. Divalent cation specificity of Q. thermophila acetate kinase. Metal ion Activitya Mg2+ 100 Mn2+ 130 Co2+ 30 002+ 30 002+ NSb 1112+ NS Zn2+ NS aThe hydroxamate assay was used as described in the text except where the chloride salts of the indicated cations (10 mM) replaced MgCl2. The reactions were initiated with 0.12 ug of enzyme. Activity with Mg2+ (1002) was 518 umoles acetyl phosphate/min per mg protein. bNS: No significant activity. 1 1 1 1 — 1 ¤I 66 I E z·xE •/"nI\ \DS 40 - I \„/ ‘ ; 30 . O I gg·< 20 / · 20 I IEILJ U) 10 •’•/ 4.0 5.0 6.0 7.0 8.0 Figure 16. Influence of pH on activity of phosphate acetyltransferase in cell extracts of M. thermophila. Assays to determine specific activity were performed as described in Materials and Methods except that CoA was at 0.2 mM and Tris buffer was replaced with a mixture of TES and MES buffers (each at 50 mM) adjusted to the desired pH with KOH (MES, (2-[N-morpholino]ethanesu1fonic acid). Assay mixtures contained 2.5 ul of 1:30 diluted (in water) cell extract containing 7 pg protein. 56 /7 ·— V:3 30 OÖ I" J ·x 20 TEIVIPERATLIRE (OC) Figure 17. Influence of temperature on activity of phosphate acetyltransferase in cell extracts of M. thermophila. Activity assays ‘were performed as described in Materials and Methods except that CoA was at 0.2 mM and assay mixtures and cuvettes were preadjusted to the desired temperature which was maintained during the assay. Cell extract (2.5 ul of 1:30 diluted extract containing 7 ug protein) was added to the reaction mix and allowed to equilibrate for 30 sec. before initiation of the assay with acetyl-phosphate. 1· I I I I 57 purified enzyme, to be stable at this range of reaction temperatures for the reaction time used: personal communication, L. Lundie). Phosphate acetyltransferase was partially purified from extracts of acetate-grown cells by anion-exchange FPLC as previously described for acetate kinase (Figure 4). Activity eluted at 0.17 - 0.25 M KCl, with the majority of activity eluting at a lower KCl concentration than acetate kinase. A recovery of 432 of the activity loaded was obtained; the most highly purified fraction, containing 322 of the recovered activity, represented an approximately 10-fold purification to a specific activity of 333 U/mg. A component of the Methanobacterium thermoautotrophicum methylreductase system, 7—mercapt0heptanoylthreonine phosphate (HS- HTP) (71), was tested as a possible physiological coenzyme in place of CoA due to a partial structural similiarity to CoA. No acetylation of HS-HTP by partially purified phosphate acetyltransferase (similiar to that described above) (Figure 18) or by cell extracts (data not shown) was detected by following disappearance of free sulfhydryls using DTNB. In contrast, acetylation of CoA was detected under identical conditions. Similiar results were determined for CoA and HS-HTP as substrates using a spectrophotometric determination of thioester bond formation (data not shown). The rate of acetylation of CoA was not HTP.affected by the presence of equimolar or double concentrations of HS- _ Regulation of acetate kinase and phosphate acetyltransferase. When M. thermo hila is presented with both acetate and methanol, 58 • • ’§ ' N ' i O 4 8 12 16 Reoction time (min) Figure 18. Activity of E. thermoghila phosphate acetyltransferase with CoA or HS-HTP as substrates. Acetylation of CoA (Q) or HS-HTP (CÖ) by partially purified phosphate acetyltransferase (5.1 mg protein/ml, 451 U/mg) was assayed by the DTNB assay. Reactions were initiated in 2 ml of assay mixture by the addition of 45 ul of 1:300 diluted (in 20 mM phosphate buffer, pH 8.0) enzyme. I 59the_ latter is the preferred growth substrate. Methanogenesis from acetate is almost completely repressed in cells grown on methanol in the presence of acetate (121). However, acetate is used as a source of carbon and reducing potential under these growth conditions (120). Thus, the regulation of acetate kinase and phosphate acetyltransferase by acetate or methanol was investigated. The specific activities of acetate kinase and phosphate acetyltransferase in extracts of cells grown on methanol in the presence of acetate were 3.9 U/mg protein and 49 U/mg protein, respectively. These activities are comparable to those observed in cells grown with acetate alone. When equal amounts of protein from extracts of acetate- or methanol—grown cells were analyzed by Western blotting, an anti- acetate kinase antibody probe strongly reacted with a protein band from extracts of acetate-grown cells with a Mr which corresponded to acetate kinase; however, no reaction with any proteins from extracts of methanol—grown cells was visible (Figure 19). If the autoradiogram was overdeveloped, methanol-grown extracts showed only a weak reaction at the acetate kinase position (data not shown). 1 V. SUMHARY AND DISCUSSION Cell-free extracts of acetate-grown Q. thermophila were found to contain high specific activities of acetate kinase and phosphate acetyltransferase. These enzymes are proposed to function in the initial activation of acetate for conversion to methane and CO2 based, in part, on the following observations: (1) Substrate level phosphorylation is not believed to occur in methanogens (81, 13) eliminating this as a possible function for acetate kinase and phosphate acetyltransferase; (2) Both enzymes were highly enriched in compared to methanol—grown cells; (3) No l extracts of acetate—grown significant acetate-CoA ligase activity was detected in cell extracts; (A) Acetate kinase and phosphate acetyltransferase, in combination, are capable of synthesizing acetyl—CoA, the proposed activated form of acetate converted to methane by this organism (100); (5) Using available data on methane production rates in acetotrophic methanogens, the calculated activity rates of acetate kinase and phosphate acetyltransferase were sufficiently high (over 260 times faster than methane production rates) as to easily provide enough activated acetate for methane production. Acetate kinase and phosphate acetyltransferase are proposed to function additionally in the activation of acetate for cell carbon, based on the detection of high activities of these enzymes in extracts of Q. thermophila grown on methanol in the presence of acetate. The observation by Zinder et al. (120) that Q. thermophila uses acetate as . 61 62 a carbon source even in the presence of a preferred methanogenic substrate such as methanol supports this conclusion. A method of regulation in which acetate induces while methanol does not repress acetate kinase and phosphate acetyltransferase activity can be postulated. In the case of acetate kinase, this induction was shown by Western blot analysis to involve regulation of protein levels. Since it has been shown that methanogenesis from acetate is essentially eliminated in the presence of a preferred substrate such as methanol (120), it is likely that other enzymes essential for catabolism of acetate to methane are repressed by methanol; for example, the CO dehydrogenase complex in Q. thermophila, which is present at higher levels in acetate- than in methanol—grown cell extracts (103). Clearly, further work is necessary to fully define the regulatory features of this organism. The acetate kinase and phosphate acetyltransferase enzymes do not appear to be complexed to each other as the activities were partially seperated by anion-exchange chromatography (Figure 4). The results of sucrose-gradient ultra-centrifugation showed that the enzymes were not intrinsically membrane—bound, although a loose association cannot be ruled out. A more definitive localization of these enzymes is needed. Other enzymatic activities assayed in extracts of acetate-grown cells include an apparent ATPase detected by the acetate-CoA ligase assay and enzyme-linked acetate kinase assay when acetate was deleted from the reaction mixtures. This activity was not separated from the acetate kinase_activity by anion-exchange FPLC, but was markedly decreased (though not totally eliminated) in the acetate kinase V A 63 preparation after ATP affinity chromatography. The evidence is thus inconclusive but suggests that the ATPase activity is distinct from the acetate kinase enzyme. The propionate kinase activity detected in cell extracts appears to be catalyzed by the acetate kinase enzyme. This conclusion is supported by the coelution of these activities from anion—exchange FPLC and the catalysis by the purified protein of propionate phosphorylation at approximately half the rate of acetate phosphorylation. Such activity has been noted in acetate kinases from other sources (2, 16, 68) and is of unknown relevance in M. thermophila; a role in carbon assimilation is possible, as several species of methanogens have been shown to assimilate propionate (26). The function of the myokinase activity activity found in extracts is unknown. Possible clues are provided in studies done by Eyzaguirre et al. (30) and Roberton and Wolfe (78). Roberton and Wolfe discovered that AMP levels and methane production are inversely related in Methanobacterium bryantii and they speculate that AMP may be involved in the regulation of methanogenesis. Eyzaguirre et al. established AMP as a product of carbon assimilation pathways in Methanobacterium thermoautotrophicum. AMP may play similiar roles in M. thermophila. In both cases, myokinase would be likely to be involved in the regulation of AMP levels. A The acetate kinase activity in extracts of M. thermophila was _ enriched by anion·exchange and ATP affinity chromatography. The preparation obtained by this procedure was pure by denaturing gelelectophoresis.That the protein was essentially homogeneous is \ 64 supported by the detection of only one N-terminus in N—terminal amino acid analysis. Thus, the resolution of two species from this preparation by isoelectric focusing and native gel electophoresis is most likely due to one of the following reasons: (i) The presence of isozymes with alike N—termini but with minor differences in total amino acid composition and charge; (ii) An enzyme population of which a fraction is phosphorylated or substrate-bound; (iii) An enzyme population of which a fraction differs in redox state. Recent evidence, determined through two-dimensional gel electrophoresis of extracts of acetate-grown N. thermophila, supports the presence of at least two forms of acetate kinase and suggests that isozymes of other enzymes may occur in this organism (Peter Jablonski, personal communication). The results of native and denaturing molecular weight determinations imply a homodimeric form of the native acetate kinase. The single N-terminus detected further suggests that the monomers may be identical or near—identical. Native monomer, homodimer, and homotetramer forms of acetate kinase from other sources have been reported (109, 16, 68), with subunit molecular weights ranging from 40,000 to 50,000 (68, 44, 109). The presence of an essential sulfhydryl group, indicated in other acetate kinases (80, 18), is suggested in the N. thermophila enzyme by the activating and stabilizing effect of the reducing agent dithiothreitol. Activity was optimal with a Mg2+/ATP ratio near 1.0, suggesting that Mg2+ is necessary only to complex with ATP and is not required additionally for the function or stability of the enzyme. In contrast, N _ 65 a Mg2+/ATP ratio of 2.0 is optimal for the §. ggli enzyme (4). The cooperative binding of Mg2+/ATP commonly occurring in acetate kinases (16, 68, 83) was not exhibited by the Q. thermophila enzyme, as determined by the hyperbolic saturation curve for that substrate. Future study of the acetate kinase and phosphate acetyltransferase of Q. thermophila should include the following: (1) A more definitive localization of the enzymes in the cell, a study that is being undertaken by other researchers using the antibodies developed in this work; (2) Isolation of the genes coding for the enzymes and study of organization and regulation at the molecular level. The N-terminal sequence of acetate kinase determined is this study has been used by other workers to construct a probe for this purpose; (3) Further purification and characterization of phosphate acetyltransferase, a work which is in progress (L. Lundie, manuscript in preparation); (4) Study of the mechanism of catalysis of the acetate kinase, including the possibility of a covalent phosphoenzyme intermediate as determined in the E. ggli enzyme (4, 76), the possible effects on mechanism of functional differences between acetate kinases from different sources, and the possible effects on mechanism of evolutionary differences between an archaeobacterial and a eubacterial enzyme; (5) Further study of properties which contribute to thermostability. Enzymes of thermophilic organisms often lack sulfhydryl groups and contain high contents of acidic residues, although these traits have not been directly correlated with thermostability (68). 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