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Modifying the catalytic carboxylates of retaining [Beta]-glycosidases

University of British Columbia

Glycosidases hydrolyze glycosidic bonds either with retention or inversion of anomeric configuration, the mechanism employed being dictated, in part, by the distance between the two key active site carboxylates. For retaining glycosidases, the average distance is 4.5-5.5 A, while for inverting glycosidases it is greater (9-9.5 A). In the retaining endo-Po 1,4-xylanase from Bacillus circulans/subtilis, this critical distance (5.5 A) has been altered by replacing the active site nucleophile Glu78 with both a shortened and a lengthened analogue (aspartic acid and S-carboxymethyl cysteine, respectively). Shortening the nucleophilic side chain decreased kcat / Km values at least 1600 fold for the aryl P-xylobiosides. In contrast, increasing the length (achieved by selective carboxymethylation of Cys78 of the Glu78Cys mutant) reduced these values by only 16 to 100 fold. These rate differences were not reflected in the degree of bond cleavage or proton donation at the glycosylation transition state, as demonstrated by similar B1g values (Br0nsted slopes). These results confirm the importance of precise positioning of the catalytic nucleophile at the active site of B. circulans/subtilis xylanase. The acid/base catalyst of B. circulans/subtilis xylanase, Glu 172, was substituted with a glutamine, a group with no significant capacity as a proton donor/acceptor. Removal of the carboxyl side chain eliminated activity with the natural substrate xylan, though some activity, which could be further rescued in the presence of the alternate nucleophile azide, was seen with activated substrates such as the nitrophenyl xylobiosides. In addition, Glul72 was also replaced with a shortened (aspartic acid) and lengthened (S-carboxymethyl cysteine) analogue. Both shortening and lengthening this carboxyl side chain had similar effects on xylan hydrolysis, with the kcat / K m values being reduced -1000 fold relative to native xylanase. Modifying the length of the acid/base catalyst was less detrimental to the hydrolysis of aryl [3-xylobiosides. For these synthetic substrates, the kcat / Km values were decreased only 3 to 24 fold. Again, no significant change was observed in the f3ig values, suggesting that these modifications have not seriously affected the degree of bond cleavage or proton donation at the glycosylation transition state. Thus, the precise placement of the acid/base catalyst is not as critical for the hydrolysis of aryl (3-xylobiosides. It has been suggested that a cysteine could fulfill the role of the active site nucleophile in retaining glycosidases (Hardy & Poteete (1991), Biochemistry 30, 9457). To test the validity of this proposal, a kinetic evaluation was conducted on the active site nucleophile cysteine mutants of two retaining 13-glycosidases. In the case of B. circulans xylanase, the cysteine mutant (Glu78Cys) was completely inactive, not even capable of undergoing the first step (glycosylation) of the double displacement mechanism. In contrast, the corresponding cysteine mutant (Glu358Cys) of Agrobacterium (3-glucosidase did complete the glycosylation step, but the rate constant for this step was reduced at least 2 x 106 fold relative to the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu358 with a cysteine (the rate constant for this step was depressed 107 fold). Thus, Cys358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results, it seems improbable that the role of the active site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.

Thesis/Dissertation

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2009-03-30

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http://hdl.handle.net/2429/6654

Chemistry

University of British Columbia

UBCV

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