His-391 of beta-galactosidase (Escherichia coli) promotes catalyes by strong interactions with the transition state

His-391 of beta -galactosidase (Escherichia coli) was substituted by Phe, G lu, and Lys. Homogeneous preparations of the substituted enzymes were essen tially inactive unless very rapid purifications were performed, and the ass ays were done immediately. The inactive enzymes were tetrameric, just like wild-type beta -galactosidase and their fluorescence spectra were identical to the fluorescence spectrum of wild-type enzyme. Analyses of two of the s ubstituted enzymes that were very rapidly purified to homogeneity and rapid ly assayed while they were still active (at only a few substrate concentrat ions so that the data could be rapidly obtained), showed that the kinetic v alues were very similar to the values obtained with the same enzymes that w ere only partially purified. This showed that the kinetics were not affecte d by the degree of purity and allowed kinetic analyses with partially purif ied enzymes so that large numbers of points could be used for accuracy. The data showed that His-391 is a very important residue. It interacts strongl y with the transition state and promotes catalysis by stabilizing the trans ition state. Activation energy differences (Delta DeltaG(s)(double dagger)) , as determined by differences in the k(cat)/K-m values, indicated that sub stitutions for His-391 caused very large destabilizations (22.8-35.9 kJ/mol ) of the transition state. The importance of His-391 for transition state s tabilization was confirmed by studies that showed that transition state ana logs are very poor inhibitors of the substituted enzymes, while inhibition by substrate analogs was only affected in a small way by substituting for H is-391. The poor stabilities of the transition states caused significant de creases of the rates of the glycolytic cleavage steps (galactosylation, k(2 )). Degalactosylation (k(3)) was not decreased to the same extent.