Amino acid substitution enzyme bulky substrates

The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases II, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143), illuminate the role of this pocket in enzyme-substrate association. Site-specific mutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about 10 , and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about 10 (Fierke et o/., 1991). 

From the kinetic properties of L-aminoamidase (Table 9) it can be concluded that an additional methylene group adjacent to the C atom in the a-methyl-substituted substrate results in an increased affinity of the enzyme for this substrate. On the other hand, much lower affinities are observed for substrates with a bulky group directly adjacent to the C atom. Finally, the purified enzyme displays L-selective amino acid amide hydrolase activity toward both a-H- and a-alkyl-substituted amino acid amides. The enzyme has the lowest enantioselectivity toward alanine amide E 25). 

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