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

2001 ◽  
Vol 79 (2) ◽  
pp. 183-193 ◽  
Author(s):  
Reuben E Huber ◽  
Isabel Y Hlede ◽  
Nathan J Roth ◽  
Kyle C McKenzie ◽  
Kiran K Ghumman
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Strong Interactions ◽  
Wild Type ◽  
Substrate Analogs ◽  
Transition State Analogs ◽  
Transition State Stabilization ◽  
Large Numbers ◽  
Important Residue ◽  
Mechanism Transition

His-391 of β-galactosidase (Escherichia coli) was substituted by Phe, Glu, and Lys. Homogeneous preparations of the substituted enzymes were essentially inactive unless very rapid purifications were performed, and the assays were done immediately. The inactive enzymes were tetrameric, just like wild-type β-galactosidase and their fluorescence spectra were identical to the fluorescence spectrum of wild-type enzyme. Analyses of two of the substituted enzymes that were very rapidly purified to homogeneity and rapidly assayed while they were still active (at only a few substrate concentrations so that the data could be rapidly obtained), showed that the kinetic values were very similar to the values obtained with the same enzymes that were only partially purified. This showed that the kinetics were not affected by the degree of purity and allowed kinetic analyses with partially purified 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 strongly with the transition state and promotes catalysis by stabilizing the transition state. Activation energy differences (ΔΔGs‡), as determined by differences in the kcat/Km values, indicated that substitutions 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 stabilization was confirmed by studies that showed that transition state analogs are very poor inhibitors of the substituted enzymes, while inhibition by substrate analogs was only affected in a small way by substituting for His-391. The poor stabilities of the transition states caused significant decreases of the rates of the glycolytic cleavage steps (galactosylation, k2). Degalactosylation (k3) was not decreased to the same extent.Key words: β-galactosidase, mechanism, transition state, binding, histidine, catalysis.

Substitutions for Gly-794 show that binding interactions are important determinants of the catalytic action of β-galactosidase (Escherichia coli)

1994 ◽  
Vol 72 (7-8) ◽  
pp. 313-319 ◽  
Author(s):  
Mercedes Martinez-Bilbao ◽  
Reuben E. Huber
Keyword(s):  
Transition State ◽  
Negative Charge ◽  
Catalytic Efficiency ◽  
Similar Extent ◽  
Binding Interactions ◽  
Substrate Analogs ◽  
Transition State Analogs ◽  
Mechanism Transition ◽  
Relative Inhibition ◽  
Positively Charged

Substitutions of Gly-794 (β-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 β-galactoside and lactose) but decreased for o-nitrophenyl β-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. If planar and positively charged galactose derivatives are transition state analogs, they must mimic the transition state for galactosylation (k2) more than the transition state for degalactosylation (k3), since k2 usually increased when the relative inhibition by these inhibitors was better while k3 always decreased. The amounts of Mg2+ required for activation of the substituted enzymes did not correlate with either charge or size.Key words: β-galactosidase, binding, glycine, aspartic, asparagine, glutamic, lysine, mechanism, transition state, catalysis, site-specific substitution.

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

2001 ◽  
Vol 79 (2) ◽  
pp. 183-193 ◽  
Author(s):  
Reuben E. Huber ◽  
Isabel Y. Hlede ◽  
Nathan J. Roth ◽  
Kyle C. McKenzie ◽  
Kiran K. Ghumman
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Strong Interactions

Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli)

2010 ◽  
Vol 88 (5) ◽  
pp. 861-869 ◽  
Author(s):  
Megan L. Dugdale ◽  
Dayna L. Dymianiw ◽  
Bhawanjot K. Minhas ◽  
Igor D’Angelo ◽  
Reuben E. Huber
Keyword(s):  
Energy Level ◽  
Transition State ◽  
Conformational Changes ◽  
Open Loop ◽  
Side Chain ◽  
Transition State Analog ◽  
Substrate Analogs ◽  
Transition State Analogs ◽  
Substrate Analog

The Met-542 residue of β-galactosidase is important for the enzyme’s activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794–803). Phe-601 and Arg-599, which interact with each other via the π electrons of Phe-601 and the guanidium cation of Arg-599, move out of their normal positions and become disordered when Met-542 is replaced by an Ala residue because of the loss of the guide. Since the backbone carbonyl of Phe-601 is a ligand for Na+, the Na+ also moves out of its normal position and becomes disordered; the Na+ binds about 120 times more poorly. In turn, two other Na+ ligands, Asn-604 and Asp-201, become disordered. A substrate analog (IPTG) restored Arg-599, Phe-601, and Na+ to their normal open-loop positions, whereas a transition state analog (d-galactonolactone) restored them to their normal closed-loop positions. These compounds also restored order to Phe-601, Asn-604, Asp-201, and Na+. Binding energy was, however, necessary to restore structure and order. The Ks values of oNPG and pNPG and the competitive Ki values of substrate analogs were 90–250 times higher than with native enzyme, whereas the competitive Ki values of transition state analogs were ~3.5–10 times higher. Because of this, the E•S energy level is raised more than the E•transition state energy level and less activation energy is needed for galactosylation. The galactosylation rates (k2) of M542A–β-galactosidase therefore increase. However, the rate of degalactosylation (k3) decreased because the E•transition state complex is less stable.

Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization

Biochemistry ◽  
1992 ◽  
Vol 31 (34) ◽  
pp. 7826-7833 ◽  
Author(s):  
Luyuan Li ◽  
Peter E. Wright ◽  
Stephen J. Benkovic ◽  
Christopher J. Falzone
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Transition State Stabilization

scholarly journals Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates

2013 ◽  
Vol 42 (1) ◽  
pp. 631-642 ◽  
Author(s):  
Shiying Wu ◽  
Yu Chen ◽  
Guanzhong Mao ◽  
Stefan Trobro ◽  
Marek Kwiatkowski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Ribonuclease P ◽  
Transition State Stabilization ◽  
Ribonuclease P Rna

scholarly journals Mutants with Temperature-Sensitive Defects in the Escherichia coli Mismatch Repair System: Sensitivity to Mispairs Generated In Vivo

2005 ◽  
Vol 187 (3) ◽  
pp. 840-846 ◽  
Author(s):  
Esther S. Hong ◽  
Annie Yeung ◽  
Pauline Funchain ◽  
Malgorzata M. Slupska ◽  
Jeffrey H. Miller
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Temperature Sensitive ◽  
Repair System ◽  
Wild Type ◽  
System Sensitivity ◽  
Large Numbers ◽  
Mismatch Repair System ◽  
Homeologous Recombination

ABSTRACT We have used direct selections to generate large numbers of mutants of Escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation, mutS60, results in almost normal levels of spontaneous mutations at 37°C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in mutS at 43°C. However, at 37°C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homeologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of mutD (dnaQ), mutC, and ndk. Strains with mutS60 can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with ndk, lacking nucleotide diphosphate kinase, are striking. An ndk mutS60 strain yields four to five times the level of mutations seen in a full knockout of mutS. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.

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