Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae

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/K-serine and V/K-OAS, decrease at low pH, exhibiting a pK of similar to7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V-1/E-t, V-1/KserineEt, V/KAcCoAEt, V-2/E-t, V-2/KOASEt, and V/KCoAEt are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M-1 s(-1), 3.3 x 10(6) M-1 s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M-1 s(-1), and (4.2 +/- 0.7) x 10(5) M-1 s(-1), respectively. The K-i 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.