Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.
Cell‐free extracts of nitrate‐grown as well as of ammonium‐grown cells of the filamentous non‐nitrogen‐fixing cyanobacterium Phormidium laminosum (strain OH‐1‐p.Cl1) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH‐dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate‐grown cells was higher than that of ammonium‐grown cells, whereas the GDH level was higher in ammonium‐grown cells and depended on the external ammonium concentration. When nitrate‐grown cells were transferred to an ammonium‐containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia‐grown cells were transferred to a nitrate‐containing medium, an increase of GS and a decrease of GDH‐specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate‐grown cells were transferred to nitrate medium with L‐methionine‐DL‐sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.
P-galactosidase (Escherichia coli ) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+. Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type ,&galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type P-galactosidase activity, the substituted enzyme has very different divalent metal interactions. A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme. The inactivation of E461H-P-galactosidase caused by Mg2+ took about 20 min. Reactivation by removal of the divalent metal took about 60 min. Interaction with Mg2+ was about IO7-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values. "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-P-galactosidase at pH values above 6 with both o-nitrophenyl P-D-galactopyranoside and p-nitrophenyl P-D-galactopyranoside in both the presence and absence of Mg2+. Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-P-galactosidase (apparent pK, was about 6.8). The substitution of Glu-461 by His, therefore, causes galactosylation to decrease, and the addition of divalent cation to the enzyme causes a further pH dependent decrease of the rate. Analysis of the results indicated that Glu-461 may function to orient and possibly stabilize an intermediate galactosyl cation and that Mg2+ might align and modulate the effect of Glu-461. The Mg2+ binding site also seems important for the overall geometry of the binding site since addition of Mg2+ to E461H-P-galactosidase caused increases of about 10-fold in the K, values.
Substitutions of Gly-794 (beta-galactosidase) with Asp, Asn, Glu, and Lys caused decreased binding of substrates and inhibition by substrate analogs, while inhibition by planar and positively charged galactose analogs increased relative to the binding of substrates and the inhibition by substrate analogs. There was a correlation of the relative inhibition with the size of the substituted residue but no relationship to the presence or absence of a negative charge, and as the relative inhibition by the planar and positively charged galactose analogs increased, k3 (hydrolysis; degalactosylation) and kcat/Km (catalytic efficiency) values decreased. The k2 values (glycolytic cleavage; galactosylation) mainly increased for poor substrates (p-nitrophenyl beta-galactoside and lactose) but decreased for o-nitrophenyl beta-galactoside (a good substrate). Enzymes substituted with Asp or Asn were inhibited to a similar extent by planar and positively charged inhibitors and had similar effects on catalysis, while inhibition and catalytic effects on the enzyme substituted by Glu were quite different. If the negative charge was important, the Asp- and Glu-substituted enzymes should have been inhibited to a similar extent, while the Asn-substituted enzyme should have caused a different degree of inhibition. The enzyme substituted with a Lys at position 794 bound substrates and inhibitors very poorly, but the relative inhibition and the catalysis still correlated to size. Alterations of the size of the residue at position 794 cause modifications in the binding interactions and affected activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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