Mechanism of adenylate kinase. Critical evaluation of the X-ray model and assignment of the AMP site
- The Ohio State Univ., Columbus (USA)
The substrate binding sites of adenylate kinase (AK) proposed by X-ray crystallographic studies were evaluated by site-specific mutagenesis in conjunction with structural analysis by NMR. The residues examined in this report include two near an adenosine site (threonine-39 and arginine-44) and two in the phosphate binding region (arginine-128 and arginine-149). The results and conclusions are summarized as follows: (a) Although Thr-39 is very close to an adenine site, it is nonessential either structurally or functionally. (b) The R44M mutant enzyme showed significant increases in the Michaelis and dissociation constants of adenosine 5{prime}-monophosphate (AMP) while all other kinetic parameters were relatively unperturbed. These results indicate that Arg-44 interacts specifically with AMP starting at the binary complex, and suggest that the MgATP site proposed by Pai et al. (1977) is likely to be the AMP site. (c) The kinetic parameters of R149M were dramatically perturbed. These results led to the conclusion that Arg-149 stabilizes the ternary. (d) The k{sub cat}, K{sub m}, and dissociation constant of R128A were all perturbed. (e) One set of AP{sub 5}A resonances was assigned to the AMP site on the basis of two findings. (f) The results are used to critically evaluate existing binding site models, including the most recent addition by Kim et al.
- OSTI ID:
- 5613546
- Journal Information:
- Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:49; ISSN 0006-2960; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model
Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine
Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
ADENYLIC ACID
AMINO ACIDS
AMP
ARGININE
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CATALYSIS
COHERENT SCATTERING
DIFFRACTION
ENZYMES
HYDROXY ACIDS
KINETICS
MAGNETIC RESONANCE
MEMBRANE PROTEINS
MUTAGENESIS
NUCLEAR MAGNETIC RESONANCE
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
REACTION KINETICS
RECEPTORS
RESONANCE
SCATTERING
STRUCTURAL MODELS
SUBSTRATES
THREONINE
TRANSFERASES
X-RAY DIFFRACTION