Chemical Mechanism of the Serine Acetyltransferase from Haemophilus influenzae

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Abstract
The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid−base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/Kserine and V/KOAS, decrease at low pH, exhibiting a pK of ∼7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V1/Et, V1/KserineEt, V/KAcCoAEt, V2/Et, V2/KOASEt, and V/KCoAEt are 3300 ± 180 s-1, (9.6 ± 0.4) × 105 M-1 s-1, 3.3 × 106 M-1 s-1, 420 ± 50 s-1, (2.1 ± 0.5) × 104 M-1 s-1, and (4.2 ± 0.7) × 105 M-1 s-1, respectively. The Ki values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 ± 0.2 and 2.5 ± 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.