Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

Journal Article · Tue Jun 14 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00412a052· OSTI ID:7163450

Cronin, C N; Kirsch, J F

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of asparate aminotransferase. It forms a salt bridge with the ..beta.. or ..gamma.. carboxylate group of the substrate. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity k/sub cat//K/sub M/) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and ..cap alpha..-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9-fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (k/sub cat//K/sub M/)/sub cationic//(k/sub cat//K/sub M/)/sub anionic/ is in the range 2-40-fold for R292D, whereas this ratio has a range of ((0.3-6) x 10/sup -6/)-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity. Possible explanations for the less-than-expected reactivity of R292D with arginine are discussed.

• Research Organization: Lawrence Berkeley Lab., CA (USA)
• DOE Contract Number: AC03-76SF00098
• OSTI ID: 7163450
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:12; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550602* -- Medicine-- External Radiation in Diagnostics-- (1980-)
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
AMINOTRANSFERASES
ARGININE
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
COHERENT SCATTERING
CRYSTALLOGRAPHY
DIFFRACTION
ELECTROPHORESIS
ENZYMES
KINETICS
LYSINE
MUTAGENESIS
NITROGEN TRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORNITHINE
REACTION KINETICS
SCATTERING
SUBSTRATES
TRANSFERASES
X-RAY DIFFRACTION