Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Biochemistry ◽  
1990 ◽  
Vol 29 (24) ◽  
pp. 5790-5796 ◽  
Author(s):  
Maria A. Vanoni ◽  
Kenny K. Wong ◽  
David P. Ballou ◽  
John S. Blanchard
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Glutathione Reductase ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Rapid Reaction

scholarly journals Investigation of α-deuterium kinetic isotope effects on the purine nucleoside phosphorylase reaction by the equilibrium-perturbation technique

1989 ◽  
Vol 257 (2) ◽  
pp. 355-359 ◽  
Author(s):  
P K Lehikoinen ◽  
M L Sinnott ◽  
T A Krenitsky
Keyword(s):  
Escherichia Coli ◽  
Equilibrium Constant ◽  
Purine Nucleoside Phosphorylase ◽  
Perturbation Technique ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Kinetic Isotope

1. alpha-Deuterium kinetic isotope effects on the phosphorolysis of inosine catalysed by Escherichia coli purine nucleoside phosphorylase were measured by the equilibrium-perturbation technique, by using the change in absorbance at 250 nm (approx. 20%). 2. Values of 2H(V/K) of 1.13(9) at pH 5.0, 1.10(5) at pH 6.1, 1.09(4) at pH 7.3, 1.08 at pH 8.4 and 1.16(4) at pH 9.4 were obtained. 3. These are compared with literature alpha-deuterium kinetic isotope effects for this and related reactions. 4. The equilibrium constant, defined as [inosine].[H2PO4-]/[hypoxanthine] [alpha-Rib f OPO3H-], is 46 at 25 degrees C. 5. N-3-beta-D-Ribofuranosylhypoxanthine, an impurity in chemically synthesized inosine, is a substrate.

scholarly journals Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase

2005 ◽  
Vol 388 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Parvinder HOTHI ◽  
Michael J. SUTCLIFFE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Steady State ◽  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Competitive Binding ◽  
Catalytic Site ◽  
Methanol Dehydrogenase ◽  
Ammonium Concentration ◽  
Steric Interaction ◽  
Kinetic Isotope

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing ‘endogenous’ activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966–3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI′). The KI′ site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.

Substrate kinetic isotope effects in dehydrogenase coupled active transport in membrane visicles in Escherichia coli

Biochemistry ◽  
1977 ◽  
Vol 16 (12) ◽  
pp. 2619-2628 ◽  
Author(s):  
Gregory J. Kaczorowski ◽  
Yak-Fa Cheung ◽  
Christopher Walsh
Keyword(s):  
Escherichia Coli ◽  
Active Transport ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope
Sign in / Sign up

Export Citation Format

Share Document