The role of Leu-190 in the function and stability of adenylate kinase.
To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.
Yoneya
T
T
Institute of Scientific and Industrial Research, Osaka University, Japan.
Okajima
T
T
Tagaya
M
M
Tanizawa
K
K
Fukui
T
T
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D007930:Leucine