Altering the Structure of Enzymes by Site-Directed Mutagenesis
Site-directed mutagenesis of enzymes allows allows the direct study of the contributions of specific side-chains to substrate binding and catalysis. This has led to a breakthrough in understanding relationships between structure and function. Tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus has been a paradigm for protein engineering studies on structure reactivity relationships in catalysis. Kinetic analysis of TyrTS mutants has given quantitative information which can be applied to many proteins. The interaction energy between the enzyme and its substrates during catalysis has been determined, allowing measurement of the apparent strengths of hydrogen bonds and salt bridges. It has been shown directly that catalysis results from stabilization of the transition state. The gross structure of TyrTS has also been investigated. The enzyme comprises 2 subunits of identical composition, and each monomer has 2 discrete functional domains: one which binds tRNA and the other which contains the active site. tRNA interacts with both subunits, binding to one and then being charged at the other subunit of the dimer. Each monomer has a complete active site, but only 1 site in each dimer functions catalytically. TyrTS is thus a classical example of an enzyme with half-of-the-sites activity. The mechanism of TyrTS has been studied and it has been shown that each dimer uses the same active site repeatedly. The second subunit has no detectable activity so that the enzyme has long-lasting asymmetry in function. Asymmetry is an inherent property and is not induced by binding of substrate. This accounts for half-of-the-sites activity and shows that the enzyme has an asymmetrical structure in solution, contrasting with the structure in crystals which is symmetrical about the subunit interface. A monomer of the enzyme is probably too small to allow both recognition and charging of tRNA, explaining the requirement for the enzyme to function as an asymmetric dimer. The enzyme appears to bind two molecules of Tyr sequentially to the same site during charging of 1 molecule of tRNA. The second molecule of Tyr perhaps aids the dissociation of charged tRNA by displacing the tyrosyl moiety from its binding site.
KeywordsProtein Engineering Mutant Enzyme Subunit Interface Biphasic Kinetic Crystalline Enzyme
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