A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

Abstract

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

“…We typically define residues in contact with the substrate as first shell, those in contact with the first shell as second shell and those in contact with the second shell as third shell. Certain second-shell residues have been shown to have a profound impact on PGI function, while distal residues contribute little to the activity of KSI [Somarowthu et al, 2011a], as predicted. Therefore, changing residues that are distally located from the active site can impact different enzymes differently.…”

“…The specificity scores achieved by THEMATICS [Wei et al, 2007] and POOL [Tong et al, 2009;Somarowthu et al, 2011b] are substantially higher than those obtained from simple sequence conservation-based methods. Indeed, as was demonstrated in earlier studies of NH [Brodkin et al, 2011] and of KSI and PGI [Somarowthu et al, 2011a], most of the residues predicted by THEMATICS prove to be important for catalysis, whereas negative control residues, i.e., conserved nearby residues that are not predicted by THEMATICS, are shown not to play a significant role in catalytic efficiency.…”

“…In this work, we focused on the residues ranked in the top 10 of the predicted residues. These functional site predictors have been used to correctly predict important residues outside of the first shell in several different enzymes [Brodkin et al, 2011;Somarowthu et al, 2011a]. In each of the previous cases, the substrates were small molecules.…”

“…DOI 10.1002/em the active site residues through steric, pi systems, or electrostatic interactions that are either lost or reduced in the mutations made. Identifying these distal residues and characterizing their contributions to activity may lead to a better understanding of the enzymes as well as to controlled manipulation of enzyme activity and specificity [Somarowthu et al, 2011a].…”

“…Such distal residues, although they do not come into direct contact with the substrate, may influence specificity or efficiency, possibly by their contact with those amino acid residues that do contact the substrate. These predictions of participation in catalysis by distal residues has been verified experimentally for nitrile hydratase (NH) [Brodkin et al, 2011], phosphoglucose isomerase (PGI) and ketosteroid isomerase (KSI) [Somarowthu et al, 2011a]. We typically define residues in contact with the substrate as first shell, those in contact with the first shell as second shell and those in contact with the second shell as third shell.…”