Reduced activity of BamHI variants C54I, C64W, and C54D/C64R is consistentwith the substrate-assisted catalysis model

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restri ction endonuclease BamHI, were generated and studied to investigate the rol e, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI . The mutation was achieved using the megaprimer approach for PCR. The muta nt genes were cloned and characterized by sequencing. The mutant and the wi ld-type proteins were expressed and purified and their kinetic parameters w ere determined using short synthetic oligonucleotides as substrates. All mu tants had higher K-m values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protei n C54W showed significant changes in the CD spectra vis-a-vis wild-type enz yme and had the lowest K-m/K-cat value among the mutants indicative of chan ges in the secondary structure of the protein. The melting curves of the mu tant proteins overlapped that of the wild-type enzyme. Analysis of the K-ca t values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substr ate-assisted catalysis mechanism. (C) 2001 Academic Press.