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

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.

ChemInform Abstract: A Potent Transition-State Analogue Inhibitor of Escherichia coli Asparagine Synthetase A.

ChemInform ◽  
2010 ◽  
Vol 30 (39) ◽  
pp. no-no
Author(s):  
Mitsuteru Koizumi ◽  
Jun Hiratake ◽  
Toru Nakatsu ◽  
Hiroaki Kato ◽  
Jun'ichi Oda
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Asparagine Synthetase ◽  
Transition State Analogue

scholarly journals Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) β-galactosidase

1992 ◽  
Vol 286 (3) ◽  
pp. 721-727 ◽  
Author(s):  
J D McCarter ◽  
M J Adam ◽  
S G Withers
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Kinetic Parameters ◽  
Parent Compound ◽  
Order Rate Constant ◽  
Linear Free Energy Relationship ◽  
Electronic Effects ◽  
Linear Free Energy ◽  
Covalent Intermediate ◽  
Hydrolysis Of

Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2′,4′-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.

Probes of the structure of phosphoenolpyruvate synthetase: Effects of a transition state analogue on enzyme conformation

1978 ◽  
Vol 56 (8) ◽  
pp. 816-819 ◽  
Author(s):  
Suree Narindrasorasak ◽  
William A. Bridger
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transition State ◽  
Enzymatic Reaction ◽  
Inhibition Constant ◽  
Amino Acid Residues ◽  
Complete Inactivation ◽  
Transition State Analogue ◽  
Reactive Groups

Phosphoenolpyruvate synthetase of Escherichia coli is strongly inhibited by oxalate. The magnitude of the inhibition constant for oxalate suggests that this compound acts to produce a transition state analogue, in keeping with the suggestion of others that oxalate mimics the structure of enolpyruvate, a presumed catalytic intermediate in the enzymatic reaction. The addition of oxalate together with ATP results in a dramatic shielding of sensitive amino acid residues from reaction with both N-ethyl maleimide and phenylglyoxal. Thus, under conditions otherwise giving rise to almost complete inactivation by either reagent, no loss of activity is detectable in the presence of oxalate and ATP. These results indicate the formation of an enclosed structure during catalysis in which reactive groups are rendered quite inaccessible to solvent.

scholarly journals Catalytic consequences of experimental evolution: catalysis by a ‘third-generation’ evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a ‘second-generation’ evolvant containing two supposedly ‘kinetically silent’ mutations

1995 ◽  
Vol 312 (3) ◽  
pp. 971-977 ◽  
Author(s):  
S Krishnan ◽  
B G Hall ◽  
M L Sinnott
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Large Subunit ◽  
Small Subunit ◽  
Enzyme Hydrolysis ◽  
Kinetic Activity ◽  
Energy Profiles ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Beta Galactosidase

The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms (‘evolvants’), ebgabcd and ebgabcde, of the second beta-galactosidase of Escherichia coli have been measured. The ebgabcd enzyme differs from the wild-type (ebgo) enzyme by Asp92-->Asn (a) and Trp977-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser979-->Gly in the large subunit (c) and Glu122-->Gly in the small subunit (d). The enzyme ebgabcde contains in addition a Glu93-->Lys change in the large subunit (e). Comparison of ebgabcd with ebgab [Elliott, K, Sinnott, Smith, Bommuswamy, Guo, Hall and Zhang (1992) Biochem. J. 282, 155-164] indicates that the c and d changes in fact accelerate the hydrolysis of the glycosyl-enzyme intermediate by a factor of 2.5, and also decrease the charge on the aglycone oxygen atom at the first transition state; the charge on the glycone, however, is unaltered [see K, Konstantinidis, Sinnott and Hall (1993) Biochem. J. 291, 15-17]. The e mutation causes a fall in the degalactosylation rate of about a factor of 3, and its occurrence only together with c and d mutations [Hall, Betts and Wootton (1989) Genetics 123, 635-648] suggests that degalactosylation of a hypothetical ebgabe enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebgab. The transfer products from galactosyl-ebgabcd and galactosyl-ebgabcde to high concentrations to glucose have been measured; the predominant product is allolactose, but significant quantities of lactose are also formed; however, at apparent kinetic saturation of the galactosyl-enzyme, hydrolysis rather than transfer is the preponderant pathway. A knowledge of the rates of enzyme-catalysed exchange of 18O from [1-18O]galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by begabcd and ebgabcde. As with the other evolvants, changes in the profile away from the rate-determining transition state are essentially random, and there is no correlation between the changes in the free energies of intermediates and of their flanking transition states. We consider the aggregate of our kinetic data on the ebg system to be telling experimental support for the theoretical objections of Pettersson [Pettersson (1992) Eur. J. Biochem. 206, 289-295 and previous papers] to the Albery-Knowles theory of the evolution of enzyme kinetic activity.

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