Role of leucine 66 in the asymmetric recognition of substrates in chicken muscle adenylate kinase.
Adenylate kinase has two distinct binding sites for nucleotide substrates, MgATP and AMP. To identify the location of the site that specifically interacts with the adenine ring of AMP, we have substituted Ala, Gly, Val, Gln, and Trp for Leu66 of the recombinant chicken muscle enzyme by site-directed mutagenesis. All the purified Leu66 mutant enzymes exhibited an essentially identical circular dichroism spectrum and had thermal stabilities similar to the wild-type enzyme. Steady state kinetic analysis showed that the Leu66 mutant enzymes have significantly decreased Vmax values and markedly large Km values only for AMP. These results show that the binding site for the adenine ring of AMP in adenylate kinase is presumably located close to Leu66, which is invariant in all the enzymes so far sequenced. Significant inhibition of activities of the mutant enzymes and quenching of the Trp66 fluorescence by substrates suggest that in some Leu66 mutant enzymes, MgATP also binds to the AMP-binding site. Thus, Leu66 of adenylate kinase might play a role in the asymmetric recognition of the adenine ring of AMP from that of MgATP. Furthermore, the hydrophobicity of the residue at position 66 appears to be important for the positive cooperativity of substrate binding.
Okajima
T
T
Institute of Scientific and Industrial Research, Osaka University, Japan.
Tanizawa
K
K
Yoneya
T
T
Fukui
T
T
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D007700:Kinetics; D007930:Leucine / Q000235:genetics*; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D007930:Leucine