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 approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) 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.
Johnson
Corey M
CM
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73072, USA.
Huang
Bin
B
Roderick
Steven L
SL
Cook
Paul F
PF
D000107:Acetylation; D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004791:Enzyme Inhibitors / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006193:Haemophilus influenzae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011522:Protons; D051050:Serine O-Acetyltransferase; D012997:Solvents
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011522:Protons; D012997:Solvents; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D005998:Glycine