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331 results on '"Kagamiyama H"'

12. A large compressibility change of protein induced by a single amino acid substitution

Author

Gekko, K., Tamura, Y., Ohmae, E., Hayashi, H., Kagamiyama, H., and Ueno, H.

Subjects
Aspartic Acid, Phenylalanine, Glycine, Glutamic Acid, Proteins, Valine, Tetrahydrofolate Dehydrogenase, Pyridoxal Phosphate, Escherichia coli, Linear Models, Point Mutation, Aspartate Aminotransferases, NADP, Research Article
Abstract

The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.

Published
1996

49. Enzymatic Synthesis of 6R-[U-14C]Tetrahydrobiopterin from [U-14C]GTP

Author

Hatakeyama, K., Hoshiga, M., Suzuki, S., and Kagamiyama, H.

Abstract

An enzymatic method for preparing 6R-[U-14C]tetrahydrobiopterin from [U-14C]GTP is presented. This method utilizes purified preparations of three biosynthetic enzymes for 6R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyltetrahydropterin synthase, and rat sepiapterin reductase. Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction. The reaction conditions thereby established yielded [U-14C]biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of [U-14C]GTP. The product was subsequently isolated by high-performance liquid chromatography. The method produced labeled 6R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%.Copyright 1993, 1999 Academic Press

Published
1993
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50. Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues.

Author

Oue, S, Okamoto, A, Yano, T, and Kagamiyama, H

Abstract

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

Published
1999

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