Stopped-flow radiationless energy transfer kinetics: direct observation of enzyme-substrate complexes at steady state

Biochemistry ◽  
1980 ◽  
Vol 19 (23) ◽  
pp. 5297-5302 ◽  
Author(s):  
Roy R. Lobb ◽  
David S. Auld
Keyword(s):  
Energy Transfer ◽  
Steady State ◽  
Direct Observation ◽  
Stopped Flow ◽  
Enzyme Substrate ◽  
Radiationless Energy Transfer

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.

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 The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

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