THE ROLE OF ARGININES IN STABILIZING THE ACTIVE OPEN-LID CONFORMATIONOF RHIZOMUCOR-MIEHEI LIPASE

Molecular dynamics simulations for the lid covering the active site of Rhizomucor miehei lipase [EC 3.1.1.3] postulated that, among other in teractions, Arg86 in the lid stabilized the open-lid conformation of t he protein by multiple hydrogen bonding to the protein surface. Chemic al modification of arginine residues in R. miehei lipase with 1,2-cycl ohexanedione or phenylglyoxal resulted in residual activities in the h ydrolysis of tributyrin of 66 and 46%, respectively. Tryptic maps of n ative and phenylglyoxal-reacted R. miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the great est extent. Guanidine, a structural analog to an arginine side chain, inhibited both the native enzyme and the arginine-modified enzymes, re sulting in residual activities of 26% as compared to the native enzyme . The inhibition was not an effect of enzyme denaturation. The native enzyme was also inhibited by 1-ethylguanidine, benzamidine and urea, b ut to a lesser degree than by guanidine. Lipases from Humicola lanugin osa and porcine pancreas in 100 mM guanidine showed residual activitie s of 88 and 70%, respectively. The lipases from Candida antarctica, C. rugosa, Pseudomonas cepacia and P. fluorescens were not inhibited by guanidine. The inhibition of R. miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine r esidues suggest a role of an arginine residue in stabilizing the activ e open-lid conformation of the enzyme.