scholarly journals A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a ‘binding-change’ mechanism

1999 ◽  
Vol 341 (2) ◽  
pp. 329-337 ◽  
Author(s):  
Jamie D. VENNING ◽  
J. B JACKSON
Keyword(s):  
Steady State ◽  
Equilibrium Constant ◽  
Protein Complexes ◽  
Hydride Transfer ◽  
Order Rate Constant ◽  
Slow Phase ◽  
Single Turnover ◽  
Fast Phase ◽  
Transfer Step ◽  
Steady State Kinetics

In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (δp) to drive reduction of NADP+ by NADH. We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when [dI] < [dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which δp is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.

Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase

2002 ◽  
Vol 361 (2) ◽  
pp. 355-361 ◽  
Author(s):  
Luis M. BREDESTON ◽  
Alcides F. REGA
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Time Course ◽  
Slow Phase ◽  
Fast Phase ◽  
Lauryl Ether ◽  
Phosphorylation Reaction ◽  
Steady Level ◽  
Biexponential Kinetics ◽  
Acidic Phospholipids

Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca2+-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25°C. The time course of phosphorylation with ATP of the enzyme associated with Ca2+, probably the true phosphorylation reaction, showed a fast phase (kapp near 400s−1) followed by a slow phase (kapp = 23s−1). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (kapp = 93s−1) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (kapp = 30s−1) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (kapp = 220s−1) and slow (kapp = 20s−1) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.

scholarly journals Single-turnover and steady-state kinetics of hydrolysis of cephalosporins by β-lactamase I from Bacillus cereus

1985 ◽  
Vol 231 (1) ◽  
pp. 83-88 ◽  
Author(s):  
R Bicknell ◽  
S G Waley
Keyword(s):  
Steady State ◽  
Bacillus Cereus ◽  
Substrate Complex ◽  
Single Turnover ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Lactam Ring ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of the hydrolysis of two cephalosporins by β-lactamase I from Bacillus cereus 569/H/9 has been studied by single-turnover and steady-state methods. Single-turnover kinetics could be measured over the time scale of minutes when cephalosporin C was the substrate. The other substrate, 7-(2′,4′-dinitrophenylamino)deacetoxycephalosporanic acid, was hydrolysed even more slowly, and has potential for use in crystallographic studies of β-lactamases. Comparison of single-turnover and steady-state kinetics showed that, for both substrates, opening the β-lactam ring (i.e. acylation of the enzyme) was the rate-determining step. Thus the non-covalent enzyme-substrate complex is expected to be the intermediate observed crystallographically.

scholarly journals Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima

2003 ◽  
Vol 374 (2) ◽  
pp. 529-535 ◽  
Author(s):  
Giovanni MAGLIA ◽  
Masood H. JAVED ◽  
Rudolf K. ALLEMANN
Keyword(s):  
Steady State ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Thermotoga Maritima ◽  
Hydride Transfer ◽  
Kinetic Scheme ◽  
Transfer Rates ◽  
E Coli ◽  
Steady State Kinetics ◽  
Rate Limiting

DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 °C, which was 30 °C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s−1 at 25 °C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 °C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a pKa of 6.0. While at 30 °C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.

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