J. Biol. Chem., Vol. 265, Issue 35, 21488-21493, Dec, 1990
The role of Leu-190 in the function and stability of adenylate kinase
T Yoneya, T Okajima, M Tagaya, K Tanizawa and T Fukui
Institute of Scientific and Industrial Research, Osaka University, Japan.
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.
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