scholarly journals Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate

2007 ◽  
Vol 282 (46) ◽  
pp. 33484-33493 ◽  
Author(s):  
William C. Cooper ◽  
Yi Jin ◽  
Trevor M. Penning
Keyword(s):  
Conformational Changes ◽  
Isotope Effects ◽  
Transient State ◽  
Kinetic Mechanism ◽  
Energy Diagram ◽  
Single Turnover ◽  
Kinetic Isotope ◽  
Free Energy Diagram ◽  
Burst Kinetics ◽  
Chemical Event

Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3α-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP+ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP+ controlled kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated kcat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.

Interpretation of Transient-State Kinetic Isotope Effects†

Biochemistry ◽  
1996 ◽  
Vol 35 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Harvey F. Fisher ◽  
Swapan K. Saha
Keyword(s):  
Isotope Effects ◽  
Transient State ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
State Kinetic

scholarly journals Application of the Second Rule of Transient-State Kinetic Isotope Effects to an Enzymatic Mechanism

Biochemistry ◽  
2009 ◽  
Vol 48 (51) ◽  
pp. 12265-12271 ◽  
Author(s):  
Harvey F. Fisher ◽  
Steven J. Maniscalco ◽  
Jon Tally ◽  
Kayann Tabanor
Keyword(s):  
Isotope Effects ◽  
Transient State ◽  
Kinetic Isotope Effects ◽  
Enzymatic Mechanism ◽  
Kinetic Isotope ◽  
State Kinetic

Steady-State Enzyme Kinetics at High Pressure

1996 ◽  
Author(s):  
Dexter B. Northrop
Keyword(s):  
Transition State ◽  
Conformational Changes ◽  
Isotope Effects ◽  
Enzymatic Reaction ◽  
Kinetic Mechanism ◽  
Point Of View ◽  
Pressure Effects ◽  
Volume Changes ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Pressure effects on enzyme-catalyzed reactions were traditionally interpreted within a simplistic kinetic mechanism in which the transition state presented the highest energy barrier, and the chemical transformation of substrate to product was considered to be a singular, rate-limiting step. This was also true of isotope effects on enzyme-catalyzed reactions, but extensive isotopic studies have led to the conclusion that this transition state is rarely the highest barrier. Rather, the release of products (or the conformational change preceding product release) is usually the slowest step, often accompanied by several other partially ratelimiting steps. Thus, our interpretations of pressure effects must be shifted accordingly. Values attributed to AV‡ have been determined for more than 50 enzymes, more of them with a positive sign than negative, and most in the range of 20 to 40 ml mol–1 but these may or may not have anything to do with the activation volume associated with the transition state. Volume changes specifically associated with the binding of ligands to enzymes have been reported as well, including some very large values, as high as ΔV = 85 ml mo–1. Kinetically, these equilibrium pressure effects also originate in conformational changes because water is not very compressible; hence, rates of diffusion to and from enzymes are virtually unaffected by pressure. Much larger changes, as high as ΔV = –391 mi mo–1, have been observed during disaggregation and denaturation of enzymes. Thus, while it is possible for pressure effects to be expressed on every step of an enzymatic reaction, and to cause denaturation as well, making kinetic data from pressure effects hopelessly complex and uninterpretable, it appears likely that the most significant pressure effects will be expressed on conformational changes associated with product dissociations, without much kinetic complexity. This makes sense from another point of view—that the largest volume changes are probably on solvation equilibria during ligand binding and protein folding. Pressure effects on isotope effects have the potential of specifically identifying whether or not a volume change occurs upon attaining the transition state. With the exception of hydrogen tunneling, intrinsic isotope effects are independent of pressure.

scholarly journals Escherichia coli chorismate synthase: a deuterium kinetic-isotope effect under single-turnover and steady-state conditions shows that a flavin intermediate forms before the C-(6proR)-H bond is cleaved

1995 ◽  
Vol 305 (3) ◽  
pp. 707-710 ◽  
Author(s):  
S Bornemann ◽  
S Balasubramanian ◽  
J R Coggins ◽  
C Abell ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Bond Breaking ◽  
Chorismate Synthase ◽  
Single Turnover ◽  
Kinetic Isotope ◽  
Rate Limiting

We report the observation of a deuterium kinetic isotope effect for the conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (6proR2HV = 1.13 +/- 0.03) using recombinant chorismate synthase from Escherichia coli. Similar isotope effects were observed for the decay of a spectroscopically characterized flavin intermediate (6proR2Hk = 1.17 +/- 0.04) during single-turnover experiments. The main rate-limiting steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either before or concomitant with its decay.

Sign in / Sign up

Export Citation Format

Share Document