The pH dependence of kinetic parameters was determined in both reaction directions in order to obtain information on the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae ( HiSAT). The maximum rate in both reaction directions, as well as the V/Kserine and V/KOAS, decrease at low pH exhibiting a pK of 6--7 for a single enzyme residue that must be unprotonated for optimum activity. The pH independent values of V1/Et, V2/Et, V1/KserineEt, and V2/KOASEt, are 3270 +/- 350 s -1, 420 +/- 50 s-1, (9.6 +/- 0.4) x 105 M-1s-1, and (4.2 +/- 0.7) x 104 M-1s -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 2.8 +/- 0.2, 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. The rate-limiting step in the reaction is likely formation of the tetrahedral intermediate between serine and acetyl CoA. A brief summary of the findings and future work with HiSAT concludes the dissertation.

Enzymatic acyl transfer to alcohols and cysteine biosynthesis were reviewed in order to provide background and pose questions concerning the kinetic and chemical mechanism of serine acetyltransferase from Haemophilus influenzae . The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoA). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product and dead end inhibition patterns are all consistent with the proposed mechanism.