INSIGHTS INTO THE MECHANISM OF DOMAIN CLOSURE AND SUBSTRATE SPECIFICITY OF GLUTAMATE DEHZDROGENASE FROM Clostridium symbiosum

Antonio Miguel B. Migueis¹, Timothy J. Stillman¹, Xing-Guo Wang², Patrick J. Baker¹, K. Linda Britton¹, Paul C. Engel² and David W. Rice1

¹The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K.

²Department of Biochemistry, University College Dublin, Belfield, Dublin 4, EIRE.

Comparisons of the crystal structures of glutamate (GluDH) and leucine (LeuDH) dehydrogenase have suggested that two substitutions deep within the amino acid binding pockets of these homologous enzymes from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. To test this proposal one of these residues, K89 which hydrogen bonds to the -carboxyl group of the substrate L-glutamate in GluDH, was altered by site-directed mutagenesis to leucine. Kinetic studies have revealed that the mutant shows increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight chain amino acids to be used as substrates.

The three dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change was triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation to that of the wild type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a function of the wild type enzyme even in the absence of substrate.