Abstract:
:Tyr-503 of beta-galactosidase was specifically replaced with Phe, His, Cys, and Lys using site-directed mutagenesis. The normal enzyme and the substituted enzymes were purified. The activities of each of the substituted enzymes with o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyronoside (PNPG) were very low and Y503K-beta-galactosidase was essentially inactive, showing that Tyr-503 is important for activity. The stability (including tetrameric stability) of the enzymes at 4 and 25 degrees C was essentially the same as that of the wild-type enzyme and the cleavage patterns on sodium dodecyl sulfate gels after protease action were unchanged. These studies thus indicate that Tyr-503 has no noticeable influence on stability under normal conditions. The substitutions for Tyr-503 had some small effects on the binding of both substrate and inhibitor. However, both kappa 2 (glycosidic bond cleavage rate) and kappa 3 (hydrolysis rate constant) were dramatically reduced. Each substitution except that of Lys (which can be explained by electrostatic effects) gave decreases in kappa 2 and kappa 3 of roughly the same magnitude regardless of whether the substitutions were conservative or not. This strongly implies that the changes in rate were not due to conformational changes as it is very unlikely that there would be such similar decreases in the values of kappa 2 and kappa 3 for amino acids with such different structures and chemical properties if the changes in rate were due to conformational differences. The data suggest that one possible role of Tyr-503 is as a general acid/base catalyst. Profiles of the kinetic data of the enzymes as functions of pH supported the suggestion that Tyr-503 normally acts as a general acid and base catalyst. When Tyr-503 was substituted by His, a small amount of base catalytic activity seemed to be restored. The strongest evidence that Tyr-503 acts as an acid catalyst came from studies with isoquinolinium-beta-D-galactopyranoside as the substrate. The kappa cat(s) of Y503F-beta-galactosidase and of Y503C-beta-galactosidase decreased by about an order of magnitude while the rate decreases were about 3 orders of magnitude with ONPG and PNPG. The breakdown of isoquinolinium-beta-D-galactopyranoside cannot be catalyzed by acids.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Ring M,Huber REdoi
10.1016/0003-9861(90)90652-fsubject
Has Abstractpub_date
1990-12-01 00:00:00pages
342-50issue
2eissn
0003-9861issn
1096-0384pii
0003-9861(90)90652-Fjournal_volume
283pub_type
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