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Activation of a Covalent Enzyme-Substrate Bond By Proc. Nat. Acad. Sci. USA Vol. 70, No. 7, pp. 2077-2081, July 1973 Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector (NAD binding/structural asymmetry/acyl glyceraldehyde 3-phosphate dehydrogenase/ negative cooperativity) 0. P. MALHOTRA* AND SIDNEY A. BERNHARDt Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Ore. 97403 Communicated by V. Boekelheide, April 9, 1973 ABSTRACT The absorption spectrum of an active- reaction pathway (4). The subsequent chemical transforma- site specific chromophoric acyl enzyme, sturgeon 3-(2- tion of such intermediates is often the rate-limiting step in furyl) - acryloyl- glyceraldehyde - 3-phosphate dehydroge- nase, is reported. This acyl enzyme undergoes all of the catalysis. An example, par excellence, of the formation of a catalyzed reactions characteristic of the intermediate of stoichiometrically significant covalent intermediate is for the physiological acyl enzyme, 3-phospho-D-glyceroyl- muscle glyceraldehyde-3-phosphate dehydrogenase (GPDH; glyceraldehyde-3-phosphate dehydrogenease. The rates of EC 1.2.1.12), a tetrameric molecule composed of identical sub- reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the units. In this case an acyl (3-phosphoglycerol-) enzyme can be oxidized coenzyme, NAD+, even where the "redox" isolated in stoichiometrically significant yield (5, 6). Con- properties of the coenzyme are not required. Likewise, sistent with this finding are kinetic inferences that the rate- the spectral properties of chromophoric acyl enzyme limiting step in the oxidative phosphorylation catalyzed by are affected by the extent of bound NAD. Under the pseu- muscle GPDH (Eq. 1) near neutral pH is the of dophysiological conditions reported herein, there is a phosphorolysis stoichiometric limitation of two furylacryloyl-acyl groups the acyl enzyme (7). For such oligomeric enzymes, particularly per enzyme molecule containing four covalently-equiva- when rate-control occurs subsequent to intermediate forma- lent subunits. The binding of NAD both to the apoenzyme tion, we should consider the effect of allosteric effectors not and to the diacyl enzyme is heterogeneous: at low ex- only on the unsubstituted enzyme, but on the stoichiometri- tents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by cally significant enzyme-substrate covalent intermediates. an enzyme model involving a tetramer with 2-fold sym- 0 0 0 metry, and consequently containing equal numbers of two 1I NADI 11 HPOje V classes of sites. NAD binding to difurylacryloyl-enzyme R-C-H + EH _ R-C-E _ ' R-COP32 + EH occurs virtually discretely, first to the two unmodified n-Glyceraldehyde NADH Acyl 3-Phospho- (tight-binding) sites, followed by looser binding to the two enzyme D-glyceroyl acyl-sites. NAD occupancy at these latter sites transforms 3-phosphate HAsO}@ phosphate the chromophoric acyl spectrum from that characteristic H20 of a model furylacryloyl-thiol ester in H20 to a highly per- RCOO0 + EH turbed furylacryloyl spectrum characteristic ofmonomeric [1] native "active-thiol" furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent We have reported the isolation and spectrophotometric of NAD bound to the loose sites. Elimination of the tight identification of a chromophoric acyl enzyme with properties binding of NAD to the difurylacryloyl enzyme tetramer by analogous to that of the physiologically isolable 3-phospho- alkylation of the remaining two free SH groups with iodo- acetate has no apparent influence on the NAD-dependent glyceroyl GPDH (8). This j#(2-furyl)-acryloyl enzyme (FA- furylacryloyl-spectral perturbation at the "two equivalent enzyme), like the physiologically important acyl enzyme, acyl sites," even though it eliminates the apparent "nega- undergoes phosphorolysis, arsenolysis, and reduction by tive cooperativity" in NAD binding. NADH. Catalytic reactions involving acyl enzyme exhibit an absolute requirement for bound NAD even where the oxidative Models for allosteric activation and inhibition have centered Under primarily on the premise that this type of regulation involves potential of the coenzyme is not required (8-11). variation in the effectiveness of ligand binding (1-3). Accord- appropriate conditions, acyl enzymes can be obtained with ing to these models, allosteric effectors function by regulating stoichiometric Emits of acylation of 2 or of 4 acyl groups per tetramer (8, 12, 13). Due to concentration limitations in acyl the distribution among quaternary conformational states limitation (and consequently the fraction of states with high affinity for phosphate and to other factors, the stoichiometric substrates). In various enzyme-catalyzed reactions, formation of two acyls per tetramer is more stringent with FA-enzyme of covalent enzyme-substrate intermediates is essential to the than with the 3-phosphoglyceroyl-enzyme. Isolation of FA-enzyme from a large excess of acyl phosphate Abbreviations: GPDH, glyceraldehyde 3-phosphate dehydro- results in a diacyl tetramer containing two equivalents of genase; FA, j0(2-furyl)-acryloyl; FAP, jl(2-furyl)-acryloyl phos- bound NAD (8). The UV-visible absorption spectrum of this phate. acyl enzyme species is consistent with that of a model com- * On leave from Chemistry Department, Banaras Hindu Uni- pound FA-thiol ester in water, and exhibits only a small versity, Varanasi, India. change in spectrum when the enzyme protein is denatured t Address reprint requests to this author. (8, 14). In contrast, a monomeric enzyme that, like GPDH, 2077 Downloaded by guest on September 27, 2021 2078 Biochemistry: Malhotra and Bernhard Proc. Nat. Acad. Sci. USA 70 (1973) Since the native FA-GPDH is catalytically convertible to all of the products characteristic of the catalytic true substrate reactions (Eq. 1), it was surprising to us, on the basis of our past experience with chromophoric acyl enzymes, that FA- GPDH was spectrally unperturbed. A potential explanation lies in the known effect of NAD on the thermodynamic sta- bility of this acyl enzyme (8). As seen by chemical equilibrium measurements, the free energy of formation of the acyl enzyme bond in the FA-enzyme is strongly affected by the presence of bound NAD. The unperturbed acyl enzyme spectrum re- ported previously was for a species containing two or less bound NAD per tetramer. In the experiments that follow, we report on the effect of higher concentrations of NAD (concen- trations in the physiological range) on the spectral and chem- ical properties of the FA-enzyme. There is a profound effect of noncovalently bound NAD on the bonding properties of the acyl enzyme thiol ester, as indicated by changes in the FA absorption spectrum and the reactivity of the acyl-enzyme bond. MATERIALS AND METHODS The procedure of Allison and Kaplan (19) for purification of sturgeon muscle GPDH was modified, yielding a higher .02 specific activity. The DEAE-cellulose chromatography step was replaced by a DEAE-Sephadex chromatography step and was followed by an additional step involving carboxy methyl- .0 cellulose chromatography. The activity was estimated accord- (D>a0 ing to Ferdinand (20) and protein concentration was obtained .02 from absorbance at 280 and 260 nm. The specific activity of our enzyme preparation was 310-320 Ferdinand units/mg protein, the highest muscle GPDH activity reported. The .041 reaction of FA-phosphate (FAP) with enzyme was done as described (8). The molecular weight of the enzyme was as- sumed to be 1.4 X 105. FA-apoenzyme was prepared by passing a solution of the FA-enzyme through a small column of 310 340 370 400 Wavelength, A(nm) charcoal [washed according to Krimsky and Racker (12) ]. The 280/260 ratio of the FA-apoenzyme was in the range 2.02-2.10 FIG. 1. (a) FA-Absorption spectrum of FA-GPDH at different and was invariant to further charcoal treatments. Carboxy- concentrations of NAD. GPDH concentration is 4.2 MM and methylated FA-enzyme was prepared by treatment of the contains 1.4 FA-groups per tetramer. The NAD content varies FA-enzyme (without charcoal treatment) with a 10- to 20-fold as follows: (D) No NAD; (i) 2.4 mol of NAD per mol of GPDH; 82 ,uM; (G) 1.09 mM. (b) Difference spectra: [(FA-enzyme + excess of iodoacetate solution (pH about 7), and isolated by NAD) - (FA-enzyme) - (NAD)]. FA-enzyme in (-(b is that passage through a Bio-gel (P-30) column. The 280/260 ratio at described above. NAD concentrations are (D 41 MuM; (G 82 MM; this stage was about 1.8 and increased to 2.06 on charcoal 1.09 mM. Curve (G) is the difference spectrum (1.09 mM NAD treatment. The isolated carboxymethylated FA-enzyme was -no NAD) for carboxymethylated FA-enzyme (4.2 MAM) contain- deacylated (1-2 mM arsenate, pH 7.0) and free reactive SH ing 1.2 FA-groups and 2.8 carboxymethyl groups per tetramer. groups were estimated with Ellman's reagent [5,5'-dithio-bis- (2-nitrobenzoic acid)]. The number of such groups was in- variably equal to the number of FA groups that were initially contains a cysteine sulfhydryl at the active site and functions present, indicating that the rest of the reactive SH groups (4 by formation of a native enzyme thiol ester intermediate, - number of FA groups per tetramer) had reacted with exhibits highly perturbed ("red-shifted") chromophoric acyl iodoacetate. For rate studies, the time-dependent disappear- enzyme thiol ester spectra (14, 15). The denatured chromo- ance of the FA-GPDH absorption band was monitored at phoric acyl enzyme spectra correspond to those for the chro- 360 nm after addition of 10 Al of 40 mM arsenate solution (pH mophoric thiol esters in water. Likewise, chromophoric acyl 7.0) to a solution of FA-enzyme (total volume 0.8 ml). enzyme intermediates formed with ser;ne oxygen at the active The buffered solvent used in all the experiments contained site are highly "red-shifted" relative to the corresponding 0.01 M ethylenediamine chloride-0.1 M KCl-1 mM EDTA model esters and denatured acyl enzymes (16, 17).
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