ALTERING THE MOUTH OF A HYDROPHOBIC POCKET - STRUCTURE AND KINETICS OF HUMAN CARBONIC ANHYDRASE-II MUTANTS AT RESIDUE VAL-121

作者：NAIR SK^*; CALDERONE TL; CHRISTIANSON DW; FIERKE CA

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 1991, 266(26): 17320-17325.

DOI：10.1016/s0021-9258(19)47376-6

Eleven amino acid substitutions at Val-121 of human carbonic anhydrase II including Gly, Ala, Ser, Leu, Ile, Lys, and Arg, were constructed by site-directed mutagenesis. This residue is at the mouth of the hydrophobic pocket in the enzyme active site. The CO2 hydrase activity and the p-nitrophenyl esterase activity of these CAII variants correlate with the hydrophobicity of the residue is important for catalysis. The effects of these mutations on the steady-state kinetics for CO2 hydration occur mainly in k(cat)/K(m) and K(m), consistent with involvement of this residue in CO2 association. The Val-121 --> Ala mutant, which exhibits about one-third normal CO2 hydrase activity, has been studied by x-ray crystallographic methods. No significant changes in the mutant enzyme conformation are evident relative to the wild-type enzyme. Since Val-121 is at the mouth of the hydrophobic pocket, its substitution by the methyl side chain of alanine makes the pocket mouth significantly wider than that of the wild-type enzyme. Hence, although a moderately wide (and deep) pocket is important for substrate association, a wider mouth to this pocket does not seriously compromise the catalytic approach of CO2 toward nucleophilic zinc-bound hydroxide.

• 出版日期1991-9-15