Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4348-4352, July 1984 Biochemistry An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the (lipid activation/lipid binding/hydrophobic site/conformational change/proteolytic activation) YING-YING CHANG AND JOHN E. CRONAN, JR. Department of Microbiology, University of Illinois, 131 Burrill Hall, 407 South Goodwin, Urbana, IL 61801 Communicated by Ralph S. Wolfe, April 4, 1984

ABSTRACT The pyruvate oxidase (pyruvate:ferricyto- lipid activation of an enzyme occurs in vivo and is physiolog- chrome b, , EC 1.2.2.2) of Escherichia coli is ically significant. markedly activated by phospholipids in vitro. To test the phys- iological relevance of this activation, we isolated an E. coil mu- MATERIALS AND METHODS tant producing an oxidase that is deficient in activation by (and binding to) phospholipids. The mutant oxidase could be Bacterial Strains, Media, and Extract Preparation. Strains fully activated by a specific proteolytic cleavage, indicating CY265 (pox') and YYC124 (poxB3) are isogenic derivatives that the catalytic site is normal. The mutant enzyme functions (4) of E. coli K-12 that carry a deletion of the aceEF (pyru- poorly in vivo, indicating that activation of the oxidase by vate dehydrogenase) genes. Strain YYC124 is a pyruvate ox- phospholipids plays an important physiological role. idase mutant derived from strain CY265 (4). The strains were grown in broth containing 10 mM sodium acetate at 330C, Enzyme activation by membrane phospholipids in vitro has and cell extracts were prepared as described (3, 4). In some often been observed (1, 2) and is generally ascribed a physio- cases, the cell-free extract was heated to 600C for 5 min and logical role. However, it has not been directly demonstrated then centrifuged for 2 min in an Eppendorf centrifuge (this that the lipid activation of any enzyme observed in vitro oc- treatment removed =75% of the endogenous phospholipids curs in vivo and plays a physiological role (1, 2). We have and about one-half of the protein). The oxidase is stable to begun a genetic study (3, 4) of a lipid-activated enzyme of this heat treatment (10, 11). Membrane vesicles were pre- Escherichia coli, pyruvate oxidase (pyruvate:ferricyto- pared as described by Koland et al. (12). chrome b, oxidoreductase, EC 1.2.2.2), to ascertain the rele- Purification of Pyruvate Oxidase. The strains were grown vance of the striking lipid activation of the enzyme observed in a heavily buffered rich broth medium supplemented with in vitro (5, 6) to the' physiological function of the enzyme. glycerol and 10 mM sodium acetate. Two preparations of the E. coli pyruvate oxidase is one of the most thoroughly mutant oxidase were purified from strain YYC124. The first characterized lipid-activated (1, 5, 6). The enzyme, was done with P. Porter by the method of O'Brien et al. (10) a homotetramer of a Mr 60,000 subunit, catalyzes the con- with omission ofthe affinity column and addition of a second version of pyruvate to acetate and CO2 (5, 6). Each subunit DEAE-Sepharose column step followed by velocity centrifu- carries a tightly bound molecule of flavin and a loosely gation (48 hr at 28,000 x g in a Beckman SW28 rotor) on a bound molecule of (TPP), the bind- 10%-40% (wt/vol) glycerol gradient prepared in 0.1 M sodi- ing of which requires Mg2 . In the presence of pyruvate, um phosphate (pH 5.8). The second purification was done TPP, and Mg2+, the addition of any of a large variety of with M. Recny by the procedure of Recny and Hager (11). phospholipids, neutral lipids, or synthetic detergents results The wild-type enzyme was purified (10) by P. Porter from in a dramatic stimulation of activity; the maximum velocity strain CG6, an oxidase-overproducing strain constructed by increases about 20-fold, and the concentration of pyruvate C. Grabau of this laboratory. All three enzyme preparations needed to saturate the enzyme is decreased by a factor of 10 were >90% homogeneous as assayed on NaDodSO4/poly- (5, 6). Activation is accompanied by a tight association of the acrylamide gels or by absorption spectra, and they had spe- lipid activator with the protein. The binding of lipid to the cific activities in the presence of 20 ,M NaDodSO4 of 60-86 enzyme is accompanied by alteration of various properties of x 103 units per mg of protein. It should be noted that we the protein, which suggests conformational changes (5, 6). have studied the oxidase of E. coli K-12, whereas the previ- The lipid activation of pyruvate oxidase can be mimicked ous workers studied the enzyme from E. coli W. However, by a specific limited proteolysis with any of a variety of pro- our work and recent work from the laboratories ofR. Gennis teases in the presence of and TPP (7-9). Under and L. Hager (personal communications) indicate that the K- these conditions, the Mr 60,000 subunit of the tetramer is 12 and the W enzymes are indistinguishable both physically converted to a Mr 58,000 tetrameric species that cannot be and chemically. further activated by (or bind) lipid (7-9). Proteolytic treat- The oxidase was assayed spectrophotometrically using ment in the absence of TPP and pyruvate results in enzyme Na3Fe(CN)6 as the electron acceptor (4, 7-9). Activation inactivation and cleavage to a Mr 51,000 subunit (7-9). was assayed after incubating the enzyme with pyruvate, We have recently demonstrated that the poxB gene of E. TPP, and Mg2+ and the activator for 30 min at room tem- coli encodes pyruvate oxidase (4). Crude extracts of one of perature. A unit of enzyme activity is 1 nmol of pyruvate the poxB mutants studied, poxB3, contained a normal level decarboxylated per min. The in vivo oxidation pf pyruvate of pyruvate oxidase antigen but only 15% of the normal level was assayed using early stationary-phase cells of strains of enzyme activity (4). In this paper, we show that this mu- YYC63 and YYC164, which are pfl pps derivatives 'of tant protein has a fully functional catalytic site but is inactive strains CY265 and YYC124, respectively (4). These addi- because of an alteration in lipid binding. This is evidence that tional mutations were needed to block other pyruvate-utiliz- ing reactions (3, 4). Washed cells previously grown in succi- nate/10 mM acetate/10 mM pyruvate minimal medium (4) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. Abbreviation: TPP, thiamine pyrophosphate. 4348 Downloaded by guest on October 2, 2021 Biochemistry: Chang and Cronan Proc. NatL Acad. Sci. USA 81 (1984) 4349

Table 1. Activity of wild-type and mutant oxidase under various conditions Lipid-depleted Crude extract, units extract,* units per mg per mg of protein of protein Additions Wild type Mutant Wild type Mutant None 108 21 16(183)t 4(27)t Chymotrypsin (5 ug/ml) 108 46 33 21 Chymotrypsin (10 tg/ml) 46 196 167 Chymotrypsin (20 ug/ml) 89 1% 167 Triton X-100 (1%) 256 16 Triton X-100 (1%)/chymotrypsin (10 ,g/ml) 167 *These extracts were depleted of endogenous lipid by heating followed by centrifugation. tValues in parentheses are the oxidase activities present before the heat treatment. were assayed for release of 14CO2 from [1-14C]pyruvate, es- Crude extracts of poxB3 strains contained only 15%-20% sentially as described for cell extracts (3). of the normal level of pyruvate oxidase activity, although a Electrophoresis. NaDodSO4/polyacrylamide gel electro- normal amount of oxidase protein having a normal molecular phoresis on 10% acrylamide gels was carried out as de- weight was present (4). We found that addition of chymo- scribed (4, 7, 8). Electrophoretic transfer to nitrocellulose trypsin to these crude extracts (supplemented with pyruvate, for immunoblotting was done at 0.2 A, as described by Bur- TPP, and Mg2+) resulted in a 2- to 4-fold increase in activity, nette (13) with the inclusion of 0.1% NaDodSO4 in the trans- whereas chymotrypsin treatment of extracts of wild-type fer buffer. In addition, Triton X-100 and 10% fetal bovine cells gave no increase in activity (Table 1). Moreover, im- serum were used in place of the Nonidet P-40 and bovine munoblotting these crude extracts with antibody raised serum albumin used by Burnette (13). The anti-pyruvate oxi- against purified pyruvate oxidase showed proteolytic clip- dase concentration was 0.33 mg/ml and 125I-labeled protein ping of the mutant enzyme to the Mr 58,000 subunit charac- A (the gift of R. Kranz) was added to 5 x 104 cpm/ml. teristic of proteolytic activation, whereas the wild-type en- zyme remained largely unclipped (Fig. 1). The result ob- RESULTS tained with the wild-type enzyme was expected, because Intact cells of strain YYC164 (poxB3) were previously endogenous phospholipid was present in the crude extracts, shown to be deficient in pyruvate oxidase activity by the which would activate the oxidase and also partially protect inability of growing colonies to reduce a tetrazolium indica- the enzyme from proteolysis. The contrasting behavior of tor (4). We have now quantitated this in vivo defect by mea- the mutant enzyme strongly suggested that the poxB3 oxi- suring the production of 14Co2 from [1-14C]pyruvate by in- dase was deficient in lipid binding, because the endogenous tact cells of pox' and poxB3 strains blocked in other pyru- lipid neither fully activated the enzyme nor protected the vate-utilizing pathways. We found that the activity of the protein from chymotrypsin cleavage. poxB3 mutant was 14% that of the pox' strain (which had an This implication was tested by heating the extracts to re- activity of 67 nmol of 14CO2 produced per min per mg of move most of the endogenous lipid (presumably by coagulat- cellular protein). ing small membrane vesicles). After centrifugation, the su- pernatants of heated wild-type extracts had lost >90% of the crude extract oxidase activity, but activity was completely restored by ad- lipid-depleted dition of a lipid activator such as the nonionic detergent Tri- wild x3 pox 83 ton X-100 (Table 1). In contrast, addition of Triton X-100 to pOx wild the poxB3 extracts gave little or no stimulation of activity, pox 2 3 4 5 6 7 8 9 11 12 13 14 Mr X( 1i3 [0 although chymotrypsin treatment resulted in appearance of a 60-'-- normal level of oxidase activity (Table 1). Moreover, proteo- 58'- lytic activation of the mutant enzyme by chymotrypsin was not blocked by Triton X-100 (Table 1) and was accompanied protease - Ix 2x - Ix 2x - Ix Ix Ix - Ix Ix Ix by appearance of the Mr 58,000 proteolytically activated spe- Triton- 1%2% - 1%/2% cies (Fig. 1). In contrast, the detergent protected the wild- type oxidase from cleavage (Fig. 1). These data indicate that FIG. 1. Protease treatment of wild-type and mutant extracts un- the poxB3 pyruvate oxidase is deficient in the binding of lip- der various conditions. Either crude extracts or extracts depleted of ids required for activation. lipid (by heating and centrifugation) were treated with a-chymotryp- sin in the presence of pyruvate (0.1 M) and TPP/Mg2" (0.2 mM Studies of the Purified Mutant Oxidase. The poxB3 oxidase TPP/20 mM MgCl2) plus either endogenous lipids or Triton X-100. was purified to >90% homogeneity by the same purification The treated extracts were subjected to electrophoresis in the pres- protocols used for the wild-type enzyme. The early purifica- ence of NaDodSO4, and the resolved proteins were electrophoreti- tion steps were followed by the assay after chymotrypsin ac- cally transferred to a sheet of nitrocellulose and visualized by im- tivation. After elimination of most of the bulk protein, we munoblotting. The arrows show the positions of the undegraded M, found that NaDodSO4, an activator of the wild-type enzyme 60,000 subunit and the protease-activated Mr 58,000 fragment. Left (7, 8), also activated the poxB3 oxidase. This property was lane, purified wild-type oxidase (pox) is shown. Lanes 1-3 and 4-6 then used to follow the purification (NaDodSO4 activation are crude extracts of the wild-type strain or mutant strain, respec- was ineffective early in the purification because of binding of tively. Lanes 1 and 4, untreated; lanes 2 and 5, treated with 10 jsg of Na- chymotrypsin per ml in the presence of pyruvate and TPP/Mg2+; the detergent by bulk protein). After activation by either lanes 3 and 6, as in lanes 2 and 5 except 20 yg of a-chymotrypsin per DodSO4 or chymotrypsin, the mutant enzyme had .85% of ml. Lanes 7-10 and 11-14, lipid-depleted wild-type and mutant ex- the specific activity of the wild-type enzyme, indicating that tracts, respectively. Lanes 7 and 11, untreated; lanes 8 and 12, treat- the catalytic site of the poxB3 oxidase was essentially nor- ed with 10 mg of chymotrypsin per ml; lanes 9 and 13, treated with mal. 1% Triton X-100 followed by 10 pg of chymotrypsin per ml; lanes 10 We first studied the spectrum of lipids and detergents able and 14, as in lanes 9 and 13 but containing 2% Triton X-100. to activate the mutant oxidase. Triton X-100 was almost Downloaded by guest on October 2, 2021 4350 Biochemistry: Chang and Cronan Proc. NatL Acad Sci. USA 81 (1984)

A. Wild Type C. Wild Type 200 150 100 C12 CIO Cs C6

- 50

C- B. pow 83 Mutont] D. par B3Mutant 100 50 It~~~~~%>C12~~~~~~~C _ - CI1 C6> -- .------4 -3 -2 -6 -5 -4 -3 -2 -I Log Triton X-100 (M) Log Alkyl Sulfate (M) FIG. 2. Activation of purified pyruvate oxidase preparations by Triton X-100 or by alkyl sulfates of differing chain lengths. The activations are given relative to that seen in the presence of 20 ILM NaDodSO4. A and B show activation of the wild-type (A) and mutant (B) oxidases by Triton X-100. The molecular weight ofTriton X-100 was taken to be 640 (14). The critical micelle concentration was 0.3 mM (14). C and D show activation by the sodium alkyl sulfates of the wild-type (C) or mutant (D) oxidases. 9, NaDodSO4; A, decyl sulfate; *, octyl sulfate; x, hexyl sulfate. The levels of activity seen in the absence of activator have been subtracted from the data shown. These values were 7.5% and 5% ofthe activities in the presence of 20 jLM NaDodSO4 for the wild-type and mutant oxidases, respectively. completely ineffective in activating the mutant oxidase (Fig. from the purification of phospholipids from natural sources, 2). A detergent concentration (2.2 mM) sufficient to activate we turned to pure synthetic phospholipids. the wild type >20-fold (Fig. 2A) gave a <1-fold activation of Studies with Pure Phospholipids. Sonicated dispersions of the mutant oxidase (Fig. 2B). In contrast, NaDodSO4 (Fig. diacyl phosphatidylcholines of various chain lengths are the 2D) and cetyltrimethylammonium bromide (not shown) fully most thoroughly characterized phospholipid micelles and are activated the mutant oxidase. Shorter chain-length alkyl sul- good activators of the wild-type oxidase (16). In agreement fates were also tested (Fig. 2) and were found to be poorer with previous investigators (16), we found that diacyl phos- activators of the mutant enzyme (Fig. 2D) than of the wild- phatidylcholines of chain lengths from C6 to C16 activated type oxidase (Fig. 2C) (see below). We then tested phospho- the wild-type oxidase and that those phospholipids (diC4, lipid, the physiologically relevant activator. We first tested a diC6, diC8, and diC10) having a high critical micelle concen- mixture of phospholipids extracted from E. coli cells and, in tration activated the enzyme in the monomeric state (Fig. 4). agreement with the results found using crude extracts (Table In contrast to the wild-type enzyme, the poxB3 oxidase was 1), these lipids were unable to activate the mutant oxidase poorly activated by the diC4, diC6, diC8, and diC10 phos- (data not shown) or protect the enzyme from proteolysis phatidylcholines either above or below the critical micelle (Fig. 3). However, the degree of activation of the mutant concentration of the lipids. The results with the longer chain (but not the wild-type) oxidase by these lipids varied from lengths differed in that the diC12, diC14, and diC16 phospha- preparation to preparation, suggesting that the purification tidylcholines all activated the mutant enzyme to some ex- method was sometimes introducing an artifactual activator tent; the degree of activation increasing with chain length [e.g., lysophospholipids (15)]. To avoid problems stemming (Fig. 4). Indeed, the activity of the mutant enzyme was al-

wild type pox 83 mutant BSA 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 OVA Mr x 10 3

60-.-_ .,* _- - ..... 58- mm _l_otu 51_

pyr+TPP + + + + + + - + + + + + + +

protease - + + + + + + + - + + + + + + +

activator - - TX Ec SDS 16- 10- 10- - - TX Ec SDS 16- 10- 10- PL PC PC PC PL PC PC PC

FIG. 3. Protection of the wild-type and mutant oxidases from proteolysis. A polyacrylamide gel run in the presence of NaDodSO4 and stained with Coomassie blue is shown. Each sample (30 Al) contained purified oxidase (130 jg/ml)/67 mM sodium phosphate, pH 5.8/13.3% glycerol/20 mM MgCl2. Additions to various samples were 0.1 M pyruvate, 1.67 mM TPP, chymotrypsin (0.1 mg/ml) (this high protease concentration is necessary because of inhibition of the protease by the glycerol added to stabilize the oxidase), 2.2 mM Triton X-100 (TX), E. coli phospholipids (EcPL) (0.17 mg/ml); 200 uM NaDodSO4 (SDS), 0.9 mM dipalmitoyl phosphatidylcholine (16-PC), 1.3 mM didecanoyl phosphatidylcholine (10-PC). Pyruvate and TPP were added first, followed by the lipid or detergent, and chymotrypsin was added last. After 30 min at room temperature, the samples were diluted 1:1 with sample buffer containing NaDodSO4, boiled for 2 min, and loaded onto the gel. Lanes 1-8 and 9-16 were the wild-type and mutant oxidases, respectively, whereas molecular weight standards are bovine serum albumin (BSA) and ovalbumin (OVA). The location of the uncleaved oxidase subunit (Mr, 60,000), the proteolytically activated form (Mr, 58,000), and the proteolytically inactivated form (Mr, 51,000) are shown. Downloaded by guest on October 2, 2021 Biochemistry: Chang and Cronan Proc. NatL Acad. Sci. USA 81 (1984) 4351

[l C4j ^500 / _/C12

< 100 [ B. pBar3 Mutant DCl6 I- ., /~~~~~~~~~~~I~d ~C6 501 v Cress C14 C12 7IOJ# -6 -5 -4 -3 -5 -4 -3 -2 -I Log Diacyl Phosphatidylcholine (M) FIG. 4. Activation of purified pyruvate oxidase preparation by synthetic diacyl phosphatidylcholines. Activation of wild-type (A) and mutant oxidase (B). e, Dipalmitoyl; *, dimyristoyl; *, dilauryl; o, didecanoyl; A, dioctanoyl; o, dihexanoyl; x, dibutyryl. Concentrations of the species of chain length 2 C12 are above these critical micelle concentrations, whereas the C4, C6, and C8 concentrations are below that point. The critical micelle concentration of the C10 species is -5 x 10' M (16). Other experimental conditions are as described in Fig. 2. most normal at high concentrations of diCM6 phosphatidyl- 1/3 to 1/10th as much as the wild-type oxidase (data not choline. This chain-length dependence was independent of shown). In these experiments, an artificial electron acceptor the polar head group of the phospholipid. The relative acti- (ferricyanide) was used. A second experiment tested the vations given by the diC14 and diCM6 phosphatidylethanol- ability of the mutant oxidase to transfer electrons to 02 via amines were quantitatively similar to those given by the the cellular electron transport chain (12, 17). Under these analogous phosphatidylcholine molecules (data not shown). conditions, the poxB3 oxidase had almost no activity (Fig. A parallel result was found for the diCM4 and diC16 phospha- 5). tidylglycerols, in that the diC16 species was a much better activator than the diC14 species. In this case, however, acti- DISCUSSION vation was observed at concentrations of phospholipid the choline and ethanol- The poxB3 pyruvate oxidase is specifically defective in lipid 1/10th to 1/100th those required for a normal catalytic site, as (data not shown). The more activation. The mutant oxidase has amine diacyl phospholipids the full activity seen after activation by proteolysis activation by phosphatidylglycerol was reported shown by effective or NaDodSO4. The concentrations of pyruvate and TPP previously (16) and seems related to the net negative charge by these required to saturate the mutant enzyme activity were the of vesicles of phospholipids. as those the wild-type enzyme (data not shown). The a or detergent to activate same of The ability of given phospholipid mutant oxidase was indistinguishable from a direct indication of structure of the the wild-type or mutant oxidases is that of the wild-type enzyme in regard to molecular weight, binding of the lipid to the enzyme. This was demonstrated by (M. Recny, personal communi- the ability of the various lipids to protect the oxidase from flavin absorption spectrum the specific proteolysis by chymotrypsin (Fig. 3). All of the cation), and the products of limited proteolysis. oxidase, The most straightforward molecular interpretation of the lipid activators tested protected the wild-type in oxidase is that the lipid-bind- whereas three compounds unable to activate the mutant oxi- alteration present the poxB3 diC10 ing site of the mutant enzyme interacts less strongly with dase, Triton X-100, E. coli phospholipids, and phos- lipids than the wild-type enzyme. The driving force for inter- phatidylcholine, also failed to protect the poxB3 oxidase oxidase with the activating lipid from proteolysis. Two lipids that activated the mutant oxi- action of the wild-type dase (NaDodSO4 and diC16 phosphatidylcholine) protected

both the mutant and wild-type oxidases from chymotrypsin C cleavage (Fig. 3). 160 Chain-Length Dependence of Activation. Blake and co- 0) Wild Type that the concentration of monomeric workers (16) showed 2 120- activators needed to activate the wild-type oxidase was di- C rectly proportional to the chain length of the hydrocarbon chain, a relationship indicating that hydrophobic interactions c 80 are the major driving force in lipid activation of the oxidase. Although the poxB3 mutant oxidase can be fully activated by 40 long-chain amphiphiles such as NaDodSO4 (Fig. 2) and di- 4), the shorter chain-length al- 0 C16 phosphatidylcholine (Fig. pox 83 mutant kyl sulfates (Fig. 2) and diacyl phosphatidylcholines (Fig. 4) La^______------In------ro activated the mutant oxidase much more poorly than the 2000 4000 wild-type enzyme. Our results with the wild-type enzyme parallel those of Blake et al. (16) except that our sample of Pyruvate Oxidase Added (Units) was a poor activator. sodium dodecyl sulfate FIG. 5. consumption by E. coli membrane vesicles in Interaction of the Mutant Oxidase with E. coli Membrane the presence of the wild-type or mutant pyruvate oxidase. Mem- Vesicles. We have also examined the behavior of the poxB3 brane vesicles containing -20 pmol of cytochrome b and 6 pmol of oxidase under more physiological conditions. We first exam- cytochrome d were incubated in 0.1 M sodium phosphate, pH 6.0/10 ined the ability of the mutant oxidase to be activated by mM MgCl2/0.2 M sodium pyruvate/0.1 mM TPP. Purified prepara- phospholipids present in membrane vesicles from E. coli, tions of the wild-type (e) or mutant (o) oxidases were then added, and we found that such vesicles activated the poxB3 oxidase and 02 consumption was followed using an oxygen electrode. Downloaded by guest on October 2, 2021 4352 Biochemistry: Chang and Cronan Proc. Natl. Acad Sci. USA 81 (1984)

seems primarily hydrophobic in nature (16), the free energy lipid activation of an enzyme plays a physiological role in of binding being a direct function of the hydrocarbon chain enzyme activity. Binding of lipid by the enzyme seems to length of the activator. Although this relationship can be rig- play two roles in the action of pyruvate oxidase in vivo. orously demonstrated only for monomeric activators (such First, lipid binding activates the enzyme and increases its as the short-chain phosphophatidylcholines and alkyl sul- affinity for pyruvate and TPP/Mg2+ (5). Second, ubiqui- fates), micellar activators seem to have a similar behavior none-8, which is directly reduced by the oxidase, is thought (ref. 16; Fig. 4). The behavior of the poxB3 pyruvate oxidase to be dissolved within the phospholipid bilayer (12). Thus, if is consistent with the enzyme having an activator binding the oxidase is unable to bind to phospholipid bilayers, it may site that is less hydrophobic and, hence, less able to bind be denied access to the pool of ubiquinone and have difficul- (and be activated by) lipid molecules or micelles. This is ty passing electrons to the electron transport chain. shown by the inability of the short-chain diacyl phosphati- The phenotype of the poxB3 mutant coupled with the pre- dylcholines (C4, C6) and alkyl sulfates (C6, C8) to activate vious data from other laboratories (5, 6, 12) suggest that py- the mutant enzyme to a significant extent. As the chain ruvate oxidase could be a "shuttle" enzyme. The oxidase length (hence, the strength of interaction with the oxidase) of may be primarily cytosolic, but on exposure to a high con- the diacyl phosphatidylcholines was increased, the differ- centration of pyruvate in vivo, the enzyme would bind pyru- ence in activation between the wild-type and mutant en- vate and TPP and undergo a conformational change exposing zymes decreased markedly. The behavior of the mutant en- the lipid-. The enzyme could then be associated zyme with other detergents also can be rationalized in terms with phospholipid-rich regions of the membrane, become ac- of the strength of binding to the oxidase. CTAB, a detergent tivated, and transfer electrons to the electron transport chain that, like NaDodSO4, binds tightly to the wild-type oxidase via ubiquinone dissolved in the lipid bilayer. On exhaustion (and to proteins in general), activated the mutant enzyme, of the pyruvate, the lipid-binding site would become cryptic, whereas Triton X-100, which interacts poorly with the nor- and the enzyme would dissociate from the membrane, be- mal oxidase (and does not generally bind to proteins) was an coming cytosolic. extremely poor activator of the mutant oxidase (Table 1; Fig. 2). We thank M. Miller for help in performing the experiment in Fig. 5 With both the diacylphosphatidylcholines and the alkyl and P. Porter and M. Recny for assistance in enzyme purification. sulfates, the degree of activation of the mutant enzyme with We also thank Professors R. Gennis and L. Hager for their cooper- C8 amphiphile approximates that given by the wild-type ation in this work. This work was supported by National Science the Foundation Grants PCM7925689 and PCM8308778. enzyme with the C6 activator. Blake et al. (16) have calculat- the average contribution to the binding free energy ed that 1. Gennis, R. B. & Jonas, A. (1977) Annu. Rev. Biophys. Bioeng. per mole of methylene groups is about -0.7 kcal (1 cal = 6, 195-238. 4.184 J) for the alkyl sulfates and about -0.56 kcal for the 2. McElhaney, R. N. (1982) Curr. Top. Membr. Transp. 17, 317- monomeric diacyl phosphatidylcholines. Thus, the behavior 380. of the mutant oxidase with the C6 and C8 species of these 3. Chang, Y.-Y. & Cronan, J. E., Jr. (1982) J. Bacteriol. 151, activators (Figs. 2 and 4) suggests that the binding free ener- 1279-1289. gy of the poxB3 oxidase is weaker by 1-2 kcal/mol than that 4. Chang, Y.-Y. & Cronan, J. E., Jr. (1983) J. Bacteriol. 154, of the wild-type oxidase. This magnitude of change in the 756-762. hydrophobic binding site is consistent with the poxB3 muta- 5. Mather, M., Blake, R., Koland, J., Schrock, H., Russell, P., tion being a single amino acid change. For example, the sub- O'Brien, T., Hager, L. P., Gennis, R. B. & O'Leary, M. (1982) Biophys. J. 37, 87-88. stitution of a glycine residue for a hydrophobic residue (e.g., 6. Gennis, R. B. & Hager, L. P. (1976) The Enzymes of Biologi- phenylalanine or leucine) within the hydrophobic site would cal Membranes, ed. Martonosi, A. (Plenum, New York), Vol. result in a change in the binding free energy of 2-3 kcal/mol 2, pp. 493-504. (18-20), whereas conversion of a hydrophobic residue to a 7. Russell, P., Schrock, H. & Gennis, R. B. (1977) J. Biol. Chem. charged amino acid (18-20) would give a change of >8 kcal/ 252, 7883-7887. mol (and thus essentially eliminate the hydrophobic site). It 8. Russell, P., Hager, L. P. & Gennis, R. B. (1977) J. Biol. therefore seems that the phenotype of the poxB oxidase Chem. 252, 7877-7882. could be due to a minimal change in the primary sequence of 9. Recny, M. A. & Hager, L. P. (1983) J. Biol. Chem. 258, 5189- the protein. Such alterations are not rare, because we recent- 5195. 10. O'Brien, T. A., Schrock, H. L., Russell, P., Blake, R., II, & ly isolated an independently derived mutant with properties Gennis, R. B. (1976) Biochim. Biophys. Acta 452, 13-29. (preliminary results) similar to those of the poxB3 mutant. It 11. Recny, M. A. & Hager, L. P. (1982) J. Biol. Chem. 257, should be noted that, although the physical characterization 12878-12886. of the mutant oxidase and the genetic characterizations of 12. Koland, J. G., Miller, M. J. & Gennis, R. B. (1984) Biochemis- the poxB3 mutant are consistent with the lesion being with a try 23, 445-453. single missense mutation, we have no direct evidence that 13. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. this is the case. We were unable to isolate revertants of the 14. Helenius, A., McCaslin, D. R., Fries, E. & Tanford, C. (1979) poxB3 mutants because of the subordinate role the oxidase Methods Enzymol. 63, 734-749. plays in pyruvate metabolism in vivo (3, 4). However, we 15. Cunningham, C. C. & Hager, L. P. (1971) J. Biol. Chem. 246, 1575-1582. have isolated recombinant DNA clones carrying the poxB 16. Blake, R., Hager, L. P. & Gennis, R. B. (1978) J. Biol. Chem. gene (unpublished results), and thus DNA sequencing of the 253, 1963-1971. wild-type and poxB3 genes will directly define the mutation- 17. Shaw-Goldstein, L. A., Gennis, R. B. & Walsh, C. (1978) Bio- al change. chemistry 17, 5606-5613. Our finding that a mutant unable to oxidize pyruvate in 18. Engleman, D. M. & Steitz, T. A. (1981) Cell 23, 411-422. vivo contains pyruvate oxidase that has a functional catalytic 19. Von Heijne, G. (1981) Eur. J. Biochem. 116, 419-422. site but an altered lipid binding site, is strong evidence that 20. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. Downloaded by guest on October 2, 2021