In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

Journal Article · Mon Feb 05 23:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00457a002· OSTI ID:7063059

Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi ^[1]; Takenaka, Hitoshi; Hamada, Minoru ^[2]; Kuby, S A ^[3]

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1. Osaka Univ. (Japan)
2. Miyazaki Medical College (Japan)
3. Univ. of Utah, Salt Lake City (USA)

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP{sup 2{minus}} and for AMP{sup 2{minus}} in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP{sup 2{minus}} and MgATP{sup 2{minus}}. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K{sub m,app} values for AMP{sup 2{minus}} of the mutant enzymes, the relatively small increases in the K{sub m,app} values for MgATP{sup 2{minus}}, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site.

• OSTI ID: 7063059
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:5; ISSN 0006-2960; 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
AMP
ANIMALS
ARGININE
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
COHERENT SCATTERING
CRYSTALLOGRAPHY
DAYS LIVING RADIOISOTOPES
DIFFRACTION
ELECTROPHORESIS
ENZYMES
GENE MUTATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MAN
MUTAGENESIS
MUTANTS
MUTATIONS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PRIMATES
RADIOISOTOPES
REACTION KINETICS
SCATTERING
TRANSFERASES
VERTEBRATES
X-RAY DIFFRACTION