Insights into catalytic action mechanism of Pseudomonas mendocina 3121-1 lipase

The hydrolysis of p-nitrophenyl butyrate catalyzed by Pseudomonas mendocina 3121-1 lipase was strongly affected by guanidine hydrochloride indicating the importance of Arg residue. Since loss of the activity in the presence of urea was negligible, the inactivation could not be attributed to protein denaturation. The enzyme was unaffected by p-chlormercuribenzoic acid (p-CMB), 2-mercaptoethanol and N-ethylmaleimide (NEM) at pH 7.0 indicating that Cys residue was essential neither to catalytic action nor to structural features of the lipase. The inactivation by K3Fe(CN)6 implied that another oxidizable amino acid residue could be important. The inactivation by N-ethylmaleimide at pH 9.0 indicated the involvement of His in the catalysis. Phenylmethylsulfonyl fluoride (PMSF) strongly inhibited the enzyme pointing out the essential role of Ser residue.