期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 391, 期 2, 页码 165-170出版社
ELSEVIER SCIENCE INC
DOI: 10.1006/abbi.2001.2416
关键词
glycosylasparaginase; N-4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine; beta-N-acetyl-glucosaminyl-L-asparagine analogues; incremental binding energy; autoproteolytic activation
Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N-4-(2-acetamido-2-deoxy-beta -d-glucopyranosyl)-L-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free alpha -carboxyl group and a free alpha -amino group; computational studies suggest that the alpha -amino group actively participates in the catalytic mechanism. In order to study the importance of the alpha -carboxyl group and the alpha -amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (Delta DeltaG(b)) for those analogues that were substrates was calculated The results show that the alpha -amino group may be substituted with a group of comparable size, for the alpha -amino group contributes little, if any, to the transition state binding energy of the natural substrate. The alpha -amino group position acts as an anchor in the binding site for the substrate. On the other hand, the alpha -carboxyl group is necessary for enzyme activity; removal of the alpha -carboxyl group or changing it to an alpha -carboxamide group results in no hydrolysis reaction. Also, N-acetyl-D-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the alpha -amino group in the catalytic mechanism as indicated in computational studies. (C) 2001 Academic Press.
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