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The effect of glycerol on the activity of beta-glucosidase from Botryodiplodia theobromae Pat.

ABSTRACT: 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].

SUBMITTER: Umezurike GM

PROVIDER: S-EPMC1135041 | biostudies-other | 1988 Aug

REPOSITORIES: biostudies-other

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Project description:1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

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The effect of glycerol on the activity of beta-glucosidase from Botryodiplodia theobromae Pat.

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