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An investigation of the activity and stability of caldocellum saccharolyticum β-glucosidase Stevenson, Andrew D.


The activity and stability of the thermostable Caldocellum saccharolyticum β-glucosidase has been investigated and the properties compared to the β-glucosidase derived from the mesophile Agrobacterium faecalis, with which there exists a 50% sequence similarity. Previous work on the Agrobacterium β-glucosidase has indicated that the enzyme operates via a two-step mechanism, consisting of initial cleavage of the glycosidic bond with formation of a covalent glycosyl-enzyme intermediate (glycosylation), followed by hydrolysis of this intermediate to yield free enzyme and glycose (deglycosylation). The high degree of sequence similarity between the Agrobacterium and Caldocellum enzymes, combined with the kinetic data determined herein, strongly suggests that the Caldocellum β-glucosidase operates through the same mechanism. Values of k[sub cat] and K[sub M] were determined for enzymic hydrolysis of twelve substituted phenyl-β-D-glucopyranosides with leaving group pK[sub a]'s ranging from 5.49 to 10.32. Values of log k[sub cat] shown no significant dependence upon leaving group pK[sub a], indicative of rate-limiting deglycosylation with all substrates except those with very poor leaving groups (pK[sub a] > 9.5). Substrates with poor leaving groups show a β1[sub g] of -0.2 suggesting very little cleavage of the glycosidic bond in the glycosylation transtion state. This contrasts with the Agrobacterium (β-glucosidase which shows a β1[sub g] of -0.7, indicating significant cleavage of the glycosidic bond in the transition state. There exists a good correlation between the Agrobacterium and Caldocellum β-glucosidase log k[sub cat]/K[sub M] values for these substrates, indicative of similarities in the enzymic binding site. Values of k[sub cat] and K[sub M] were determined for enzymic hydrolysis of six 4-nitrophenyl glycopyranosides. A reasonable correlation of the log k[sub cat]/K[sub M] glycoside hydrolysis exists between the two enzymes, further indicating a similar binding site structure. An activation energy barrier of 7 kcal mol⁻¹ was determined for the Caldocellum enzyme, this comparing reasonably well with a value of 9 kcal mol⁻¹ for the Agrobacterium (β-glucosidase, again indicating a similar conformation of the two enzymes.The Caldocellum β-glucosidase is significantly more stable to both thermal and chemical denaturation than the Agrobacterium enzyme, by at least 3 kcal mol⁻¹. The stability of either enzyme was found to increase when trapped as the 2-fluoroglycosyl-enzyme intermediate, this increase in stability relating to the strength of the interactions with the substrate; again this increase in stability was at least 3 kcal mol⁻¹. The Caldocellum enzyme's higher content of hydrophobic amino acids and a lower net charge, apparent from a comparison of the sequence the two enzymes, are believed to be responsible for its greater stability.

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