Identification of essential amino acids for the catalytic activity of barley β-glucosidase

Abstract

The active site of β-glucosidase of barley seeds was investigated by means of kinetic and chemical modification studies. DEPC (diethylpyro-carbonate) totally inactivates the enzyme but not in the presence of substrate. The inactivation reaction follows pseudo-first ordr kinetics with a second-order constant of 0.049 mM −1 min −1. Reversal of inactivation in the presence of hydroxylamine leads to the inference that histidyl, tyrosyl and/or seryl residues are essential for catalysis. Treatment of the enzyme with pHMB ( p-Hydroxymercuribenzoate), PMSF (phenylmethyl-sulphonyl fluoride) and NAI ( N-acetyl-imidazole), does not influence activity, thus eliminating the possibility that cysteine, serine or tyrosine participate in catalysis. Kinetic analysis of DEPC inactivation indicates that one histidyl residue per mol of protein is essential for catalysis. EEDQ (2-ethoxy-1-ethoxy-1,2-dihydroquinoline) inactivates the enzyme, but not in the presence of substrate, following pseudo-first order kinetics with a second-order constant of 0.01 mM −1 min −1. This is indicative of the involvement of residues with a carboxyl group in the catalytic activity. Further kinetic analysis of the inactivation caused by EEDQ, strongly implies that modification of a single residue of aspartate or glutamate inactivates the enzyme. The pH profile of enzyme velocity ( V max ) and efficiency constant ( V max K m ) gave apparent pK values of 3.51 and 7.56 for the enzyme-substrate complex and 3.88 and 6.38 for the free enzyme, further supporting the concept of histidyl and carboxyl residues engagement in the catalytic activity of barley β-glucosidase.