Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction.

This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of citrate synthase. When the side chains of any of the residues (H320, D375, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate states in the multistep citrate synthase reaction. H320 forms a hydrogen bond with the carbonyl of oxaloacetate and the alcohols of the citryl-coenzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, and R) have reaction profiles for which the condensation reaction is cleanly rate determining. None of these mutants can activate the carbonyl of oxaloacetate by polarization. All these mutants catalyze the necessary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthermore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate significantly more slowly than does the wild-type. D375 is the base removing the proton of acetyl-coenzyme A. D375E and D375G have greatly diminished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wild-type. For D375E, the hydrolysis of citryl-CoA is rate determining. D375G, having no side chain capable of acid-base chemistry in either the condensation or hydrolysis reactions is nearly completely devoid of activity in any of the reactions catalyzed by the wild-type. H274 hydrogen bonds to the carbonyl of acetyl-coenzyme A but also forms the back wall of the oxaloacetate-binding site. H274G cannot properly activate either oxaloacetate or acetyl-coenzyme A, and the condensation reaction is overwhelmingly rate determining. Nonetheless, hydrolysis of the intermediate is impaired. All the enzymes except H320R and H274G show kinetic cooperativity with CitCoA as substrate, indicating changes in the subunit interactions with these latter two mutants. The energetics of citrate synthase are surprisingly tightly coupled. All changes affect more than one step in the catalytic cycle. Within the condensation reaction, the intermediate of proton transfer must occupy a shallow well between transition states close in free energy so that perturbations of one have substantial effects on that of the other.

Ability of single-site mutants of citrate synthase to catalyze proton transfer from the methyl group of dethiaacetyl-coenzyme A, a non-thioester substrate analog.

The catalytic strategies of enzymes (such as citrate synthase) whose reactions require the abstraction of the alpha-proton of a carbon acid remain elusive. Citrate synthase readily catalyzes solvent proton exchange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloacetate. Because no further reaction occurs with this analog, it provides a uniquely simple probe of the roles of active site interactions on carbon acid proton transfer catalysis. In view of the high reactivity of the analog for proton transfer to the active site base, its failure to further condense with oxaloacetate to form a sulfur-less analog of citryl-coenzyme A was unexpected, although we offer several possible explanations. We have measured the rate constants for exchange, k(exch), at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues. Comparisons between the values of k(exch) are straightforward in two limits. If the rate of exchange of the transferred proton with solvent protons is rapid, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate constants for proton transfer. If the exchange of the transferred proton with protons in the bulk solution is the slow step and the equilibrium constant for proton transfer is unfavorable (as is likely), then k(exch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bulk solvent. If that exchange rate with bulk solution remains constant for a series of mutant enzymes, then k(exch) values compare the equilibrium constants for proton transfer. The importance of the acetyl-CoA site residues, H274 and D375, is confirmed with D375 again implicated as the active site base. The results with the series of oxaloacetate site mutants, H320X, strongly suggest that activation of the first substrate, oxaloacetate, through carbonyl bond polarization, not just oxaloacetate binding in the active site, is required for the enzyme to efficiently catalyze proton transfer from the methyl group of the second substrate.

Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.

The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates.

Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction.

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes.

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase.

The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.

Evidence from 13C NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase.

The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.

Interaction of a paramagnetic analogue of oxaloacetate with citrate synthase.

Electron paramagnetic resonance studies have indicated that nitrosodisulfonate binds to pig heart citrate synthase. Titration of the enzyme with nitrosodisulfonate revealed several binding sites for the probe per subunit with one site (KD approximately 0.1 mM) having a greater affinity than the others. The substrate, oxaloacetate, competed very effectively for one of the nitrosodisulfonate binding sites (KD less than 10(-2) mM) at the same time eliminating the weaker probe binding sites. Citrate and (R)- and (S)-malates also displaced the probe. Failure to resolve low- and high-field shoulder in the high gain--high modulation electron paramagnetic resonance spectra of the enzyme--nitrosodisulfonate system indicated that the bound probe was "weakly immobilized". However, the electron paramagnetic resonance spectrum of the bound probe changed to one typical of a "strongly immobilized" nitroxide upon the addition of a saturating concentration of the substrate acetyl coenzyme A (acetyl-CoA) to the enzyme--nitrosodisulfonate system, indicating the formation of a ternary acetyl-CoA-enzyme-probe complex. Titration of the acetyl-CoA saturated enzyme with the probe indicated one binding site per subunit (KD = 0.37 mM). Thus, nitrosodisulfonate may be considered as a paramagnetic analogue of oxaloacetate in its interaction with citrate synthase. These results are compared with our previous studies with this enzyme, employing a spin-labeled acyl coenzyme A (acyl-CoA) derivative [Weidman, S. W., Drysdale, G. R., & Mildvan, A. S. (1973) Biochemistry 12, 1874--1883].

The biosynthesis of methylcitrate.

Citrate synthase catalyses the formation in vitro of two diastereoisomers of methylcitrate from propionyl-CoA and oxaloacetate. The proportion of diastereoisomers produced is temperature-sensitive. In the presence of oxaloacetate and a thiol trap, the enzyme catalyses the exchange of both alpha-protons of propionyl-CoA with 2H2O. The pro-S-proton at the alpha-carbon atom is exchanged 15 times faster with 2H2O than is the pro-R-proton. The exchanges are over 1000 and 100 times faster respectively than Vmax. These results are interpreted in the light of recent reports that 2R,3S- and 2S,3S-methylcitrates are produced by the enzyme-catalysed condensation and also excreted by patients with propionic acidaemia.