Roles of histidines 154 and 189 and aspartate 139 in the active site of serine acetyltransferase from Haemophilus influenzae

Abstract

A crystal structure of serine acetyltransferase (SAT) with cysteine bound in the serine subsite of the active site shows that both H154 and H189 are within hydrogen-bonding distance to the cysteine thiol [Olsen, L. R., Huang, B., Vetting, M. W., and Roderick, S. L. (2004) Biochemistry 43, 6013 -6019]. In addition, H154 is in an apparent dyad linkage with D139. The structure suggests that H154 is the most likely catalytic general base and that H189 and D139 may also play important roles during the catalytic reaction. Site-directed mutagenesis was performed to mutate each of these three residues to Asn, one at a time. The V1/Et value of all of the single mutant enzymes decreased, with the largest decrease (approximately 1240-fold) exhibited by the H154N mutant enzyme. Mutation of both histidines, H154N/H189N, gave a V1/Et approximately 23700-fold lower than that of the wild-type enzyme. An increase in K Ser was observed for the H189N, D139N, and H154N/H189N mutant enzymes, while the H154N mutant enzyme gave an 8-fold decrease in K Ser. For all three single mutant enzymes, V1/Et and V1/K Ser Et decrease at low pH and give a pKa of about 7, while the V1/Et of the double mutant enzyme was pH independent. The solvent deuterium kinetic isotope effects on V 1 and V1/K Ser decreased compared to wild type for the H154N mutant enzyme and increased for the H189N mutant enzyme but was about the same as that of wild type for D139N and H154N/H189N. Data suggest that H154, H189, and D139 play different catalytic roles for SAT. H154 likely serves as a general base, accepting a proton from the beta-hydroxyl of serine as the tetrahedral intermediate is formed upon nucleophilic attack on the thioester carbonyl of acetyl-CoA. However, activity is not completely lost upon elimination of H154, and thus, H189 may be able to serve as a backup general base at a lower efficiency compared to H154; it also aids in binding and orienting the serine substrate. Aspartate 139, in dyad linkage with H154, likely facilitates catalysis by increasing the basicity of H154.

Figure 1

Proposed chemical mechanism of HiSAT. (1) E–AcCoA–serine complex. (2) Tetrahedral intermediate. (3) E–CoA–OAS complex.

Figure 2

Close-up of the active site of HiSAT with cysteine bound. Locations of two subunits of the trimer of HiSAT are shown in yellow and green, respectively. The dashed lines represent potential hydrogen bonds, and the numbers close to dashed lines are distances in Å. The (A) and (B) next to numbered residues indicate the subunit that contributes the residue. The figure was created using Pymol from DeLano Scientific LLC. The HiSAT structure with cysteine bound has an access number of 1SSQ in the Protein Data Bank.

Figure 3

pH dependence of V₁/E_t (A) and V₁/K_SerE_t (B) for the H154N, H189N, and H154N/H189N mutant enzymes. Points for the H154N (◆), H189N (▲), and H154N/H189N (■) mutant enzymes are experimental values, while the curves are theoretical, based on fits of the data using eq 3.

Figure 4

pH(D) dependence of V₁/E_t (A), V₁/K_SerE_t (B), and V₁/K_AcCoAE_t (C) for the D139N mutant of HiSAT. The points shown are the experimentally determined values. pH and pD dependences are presented by (▲) and (■), respectively. The curves are theoretical, based on fits of the data using eq 3 for the pH–rate profile of V₁/E_t and eq 4 for the pH–rate profile of V₁/K_AcCoAE_t. The curve for the pD dependence of V₁/K_AcCoAE_t and the pH dependence of V₁/K_SerE_t were drawn by hand.