Application of secondary .alpha.-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and .beta.-glucosidase

Biochemistry ◽  
1969 ◽  
Vol 8 (10) ◽  
pp. 4214-4221 ◽  
Author(s):  
F. W. Dahlquist ◽  
Tsafrira Rand-Meir ◽  
Michael A. Raftery
Keyword(s):  
Enzyme Catalysis ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Glycoside Hydrolysis ◽  
Beta Glucosidase

scholarly journals The effect of glycerol on the activity of β-glucosidase from Botryodiplodia theobromae Pat

1988 ◽  
Vol 254 (1) ◽  
pp. 73-76 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Rate Equations ◽  
Glycerol Concentration ◽  
Large Excess ◽  
Reaction Sequence ◽  
Botryodiplodia Theobromae ◽  
Kinetic Isotope ◽  
Beta Glucosidase ◽  
Rate Limiting

1. In the activity of the high-Mr beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. on o-nitrophenyl beta-D-glucopyranoside as substrate, both Vmax. and Km increased non-linearly with increasing concentration of glycerol, and the Vmax./Km(app.) ratio decreased non-linearly with increasing concentration of glycerol. 2. No increase in rate was observed with phenyl beta-D-glucopyranoside as substrate in the presence of up to 250 mM-glycerol, indicating that glucosylation is rate-limiting with this substrate. 3. With o-nitrophenyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrates, kappa cat. values of 793.7 s-1, 62.8 s-1 and 5.4 s-1 respectively were calculated. 4. With o-nitrophenyl beta-D-glucopyranoside and phenyl beta-D-glucopyranoside as substrate, alpha-deuterium kinetic isotope effects of 1.9 +/- 0.03 and 1.01 +/- 0.01 respectively were found; in the presence of 200 mM-glycerol the values were 1.21 +/- 0.03 and 1.02 +/- 0.01 respectively. 5. In the presence of a large excess of o-nitrophenyl beta-D-glucopyranoside [(S] = 35.7 Km), the amount of o-nitrophenol and also of the transglucosylation product formed by beta-glucosidase action increased non-linearly, whereas that of glucose formed decreased non-linearly with increasing glycerol concentration. 6. All these results were found to fit the data calculated from rate equations derived on the basis of the proposed mechanism of enzyme action involving two ion-pair intermediates and a covalent alpha-D-glucosyl-enzyme in the reaction sequence [Umezurike (1987) Biochem. J. 241, 455-462].

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