Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering

摘要: Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase contains a large binding cleft. A conserved glycine (Gly(166)), located at the bottom left, was replaced 12 nonionic amino acids cassette mutagenesis method. Mutant enzymes showed changes in toward substrates increasing size hydrophobicity. In general, catalytic efficiency (k(cat)/K(m)) small increased (up to 16 times) substitutions position 166 Exceeding optimal volume cleft ( approximately 160 A(3)), enlarging either side chain or 166, evoked precipitous drops 5000 as result steric hindrance.