Mutational Studies of an Essential Glutamate Residue in Escherichia Coli Folylpolyglutamate Synthetase with a Role in ATP Binding
Little is known about the active site residues involved in the mechanism of catalysis of FPGS. Recently the three dimensional structure of the FPGS from Lactobacillus casei was solved by X-ray crystallography(1). The enzyme consists of two domains connected by a single loop. The active site is in the cleft between the two domains. The amino terminal domain of FPGS has the same fold as is found in a broad group of mononucleotide binding proteins, including adenylate kinase, G-proteins and p21-ras(2). The crystal structure has shown that the P-loop is truncated compared with the consensus, a feature that FPGS shares with its closest structural homologue MurD (1). Adjacent to the P-loop in the FPGS active site is Glu 143, which appears to coordinate with the magnesium ion of Mg2+ATP, suggesting that this residue is involved in the catalytic mechanism of the enzyme. In E. coli FPGS, the corresponding glutamate residue is Glu146. In this report we describe mutagenesis studies of Glu146 of the E.coli FPGS. We show that the mutant protein has no catalytic activity, although it can bind folate substrates normally. The defect caused by the mutation appears to be in ATP binding, although the mutant enzyme still binds ADP. We also show that the wild type E. coli FPGS has enhanced ATP and ADP binding and the mutant enzyme has enhanced ADP binding in the presence of dihydropteroate.
KeywordsActive Site Residue Wild Type Enzyme Mutant Enzyme Equilibrium Dialysis Lactobacillus Casei
Unable to display preview. Download preview PDF.