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

Abstract

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.

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

“…Like D375G–PCS (7), D317G– Tp CS binds ligands very tightly (Table 1 and unpublished observations). The reversibility of ternary D317G– Tp CS•OAA•defhiaacetyl-CoA complex formation was assessed using CMCoA as a kinetic trap.…”

“…As expected for a mutant enzyme lacking a key active site base, D317G– Tp CS-mediated HDX and acetyl-CoA dependent turnover rates are ∼10 –5 that of wild-type Tp CS (Table 2). For comparison, the analogous D375G–PCS•OAA HDX rate is similarly reduced, to ≤1.3 × 10 –5 that of wild-type PCS (7). …”

“…PCS Asp375 has a key role in catalysis and HDX, which are linked, as demonstrated by a parallel suppression in rates with various D375X–PCS mutants (7). In addition to affecting the rates of catalysis and HDX, the corresponding mutations in Tp CS (D317G– Tp CS, D317N– Tp CS) also affect the rate of dequenching (Table 2), a phenomenon that does not occur in PCS 3…”

“…The failure to detect dethiacitryl-CoA formation from [2- 13 C]OAA by 13 C NMR in PCS solutions was puzzling and might be due to intermediate-NMR chemical shift exchange regime effects (7). However, we now believe any dethiacitryl-CoA that is formed by PCS would be below the level of detection by this relatively insensitive method.…”

“…It seems likely that no more than one equivalent of dethiacitryl-CoA is formed by Tp CS•OAA. In contrast, PCS forms a stable, unreacted PCS•OAA•dethiaacetyl-CoA complex in solution (7), a difference that can be understood by comparing free energy reaction profiles for the two CS forms presented here.…”