Direct observation of enzyme substrate complexes by stopped-flow fluorescence: Mathematical analyses

1984 ◽  
Vol 40 (11) ◽  
pp. 1197-1206 ◽  
Author(s):  
R. R. Lobb ◽  
D. S. Auld
Keyword(s):  
Direct Observation ◽  
Stopped Flow ◽  
Enzyme Substrate ◽  
Mathematical Analyses

scholarly journals Reactions of DOPA (3,4-dihydroxyphenylalanine) decarboxylase with DOPA

1979 ◽  
Vol 183 (2) ◽  
pp. 361-368 ◽  
Author(s):  
A Minelli ◽  
A T Charteris ◽  
C B Voltattorni ◽  
R A John
Keyword(s):  
Steady State ◽  
Time Scales ◽  
Direct Observation ◽  
Equilibrium Constants ◽  
Active Part ◽  
Stopped Flow ◽  
Step Rate ◽  
Multiple Reactions ◽  
Absorbing Material ◽  
Different Time Scales

The study of DOPA (3,4-dihydroxyphenylalanine) decarboxylase by steady-state methods is difficult because multiple reactions occur. The reaction with DOPA was studied at enzyme concentrations between 20 and 50 micrometer by direct observation of the bound coenzyme by using stopped-flow and conventional spectrophotometry. Four processes were observed on different time scales and three of these were attributed to stages in the decarboxylation. The fourth was attributed to an accompanying transamination that renders the enzyme inactive. It was clear that much, if not all, of the 330 nm-absorbing coenzyme present in the free enzyme plays an active part in the decarboxylation, since it is converted into 420 nm-absorbing material in the first observable step. An intermediate absorbing maximally at 390 nm is formed in a slower step. Rate and equilibrium constants have been determined and the ratio of decarboxylation to transamination was estimated to be 1200:1.

Direct Observation of Specific Interaction between Enzyme-substrate Complexes Using Colloidal Probe Atomic Force Microscopy

2004 ◽  
Vol 33 (5) ◽  
pp. 536-537 ◽  
Author(s):  
Takehiro Suzuki ◽  
Yuan-Wei Zhang ◽  
Tanetoshi Koyama ◽  
Darryl Y. Sasaki ◽  
Kazue Kurihara
Keyword(s):  
Atomic Force Microscopy ◽  
Direct Observation ◽  
Specific Interaction ◽  
Colloidal Probe ◽  
Force Microscopy ◽  
Enzyme Substrate ◽  
Atomic Force

scholarly journals Pre-steady-state kinetics of Bacillus licheniformis 1,3-1,4-beta-glucanase: evidence for a regulatory binding site

2003 ◽  
Vol 371 (3) ◽  
pp. 997-1003 ◽  
Author(s):  
Mireia ABEL ◽  
Karin IVERSEN ◽  
Antoni PLANAS ◽  
Ulla CHRISTENSEN
Keyword(s):  
Steady State ◽  
Bacillus Licheniformis ◽  
Reaction Scheme ◽  
Activation Mechanism ◽  
Stopped Flow ◽  
Beta Glucanase ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Leaving Groups ◽  
Kinetics Of

In a previous paper, we reported the first stopped-flow experiments on a Bacillus licheniformis 1,3-1,4-β-glucanase [Abel, Planas and Christensen (2001) Biochem. J. 357, 195–202]. It was shown that the pre-steady-state kinetics of the 1,3-1,4-β-glucanase using the substrate 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside may be explained by a reaction scheme involving an induced fit and the binding of two substrates as well as a second enzymic conformational change, whereas the results definitely could not be explained in terms of the simple double-displacement scheme. In the present study, we report further stopped-flow kinetic results on the glucanase using a series of low-molecular-mass substrates with various leaving groups and varying chain length. The analysis of the resulting data leads to the conclusion that the free enzyme exists in two conformations, one of which binds the substrates rather strongly in a regulatory site, before any productive interactions can take place. This corresponds to an allosteric activation mechanism. With these substrates, however, the productive enzyme–substrate species are also able to change into less active or inactive forms. This may be seen as a feedback inhibitory mechanism.

scholarly journals Kinetic Studies on the Enzyme-substrate Complex Formation of p-Hydroxybenzoate Hydroxylase by the Stopped-flow Method

1972 ◽  
Vol 27 (10) ◽  
pp. 1172-1175 ◽  
Author(s):  
Naoki Higashi ◽  
Hirohumi Shoun ◽  
Keiji Yano ◽  
Κει Arima ◽  
Keitaro Hiromi
Keyword(s):  
Complex Formation ◽  
Reduction Rate ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Apparent Velocity ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Hydroxybenzoate Hydroxylase ◽  
Anaerobic Reduction

The spectrophotometric and spectrofluorometric investigations of the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase was made by the stopped-flow technique. The apparent velocity of the formation of the enzyme-substrate complex (the velocity of the absorbance change in visible and UV regions, and the velocity of the quenching of the fluorescence intensity in the FAD moiety of the holoenzyme by the substrate) was rapid enough to explain the maximal overall velocity (72 sec-1) or the activated anaerobic reduction rate (kredmax= 200 sec-1). The results were consistent with a two-step mechanism involving a rapid bimolecular association of enzyme and substrate, and a slower follow-up unimolecular process.

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