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

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.

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
Sign in / Sign up

Export Citation Format

Share Document