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

Abstract

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 3␣-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 k cat , whereas the chemical event also contributed to this term. k cat was insensitive to [ 2 H]NADPH, whereas D k cat /K m and the D k lim (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 k cat , 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 K eq favored reduction. The kinetic Haldane and free energy diagram confirmed that K eq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction. Mammalian hydroxysteroid dehydrogenases (HSDs)3 play pivotal roles in steroid hormone biosynthesis and metabolism (1, 2). In target tissues, they regulate the amount of steroid hormone available for their cognate nuclear receptor (3-7). This is achieved by the positional and stereospecific oxidoreduction of potent steroid hormones to their corresponding inactive metabolites or vice versa. Often these interconversions are catalyzed by pairs of HSDs that function preferentially as reductases or oxidases (5-8). There are two superfamilies of HSDs, the soluble aldo-keto reductase (AKRs) (9) and the mainly membrane-associated shortchain dehydrogenase reductases (SDRs) (10). Several HSDs have become drug targets to manipulate steroid hormone action in target tissues (11-13).Four human HSDs from the AKR superfamily, AKR1C1-AKR1C4, can reduce 3-, 17-, and 20-ketosteroids in different ratios (14, 15). They catalyze the 4-pro-R hydride transfer from NADPH to the acceptor carbonyl on the steroid substrate in an ordered bi-bi reaction where NAD(P)(H) binds first and leaves last (16,17). Rat liver 3␣-HSD (AKR1C9), which shares 69% sequence identity with its human homologs, represents the best system to relate structure to function for HSDs, because crystal structures exist for the apoenzyme (E) (18), the E⅐NADP ϩ binary complex (19), and the E⅐NADP ϩ ⅐testosterone ternary complex (where testosterone is a competitive inh...

A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases

“…These appraisals indicate that NADP(H) induces Loop B to adopt a closed conformation that fastens this cofactor into the 1B). Our data agree with kinetic studies of rat liver AKR1C9 that suggest NADPH binding occurs in three steps: 1) the fast formation of a loose complex (E$NADPH) followed by two conformational changes of the enzyme leading to 2) an intermediate (E*$NADPH) and 3) to a tightly bound complex (E**$NADPH) [24]. In this context, our results support a model in which the formation of an intermediate E*$NADPH complex anchors the adenosine-2 0 -5 0 -diphosphate moiety and consequently increases flexibility of Loop B.…”

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior

“…Almost universally, the dissociation constants calculated from the microscopic rate constants significantly underestimate those obtained by fluorescence titration and steady-state kinetics. Although the source of this disparity is unknown, some difficulty in reconciling stopped-flow fluorescence data with that measured at steady state has been reported by others (Cooper et al, 2007;Jin and Penning, 2006;Ma et al, 2000;Ratnam et al, 1999). Control experiments were performed to rule out artifacts due to mixing effects, non-specific binding, or photobleaching with the fluorescence methods, and the introduction of additional steps in the cofactor binding mechanism could only further decrease the calculated dissociation constants.…”

“…The mechanism of cofactor binding in a model AKR, rat 3a-hydroxysteroid dehydrogenase (3a-HSD), has been extensively studied and demonstrates a multi-step binding mechanism for the NADP(H) cofactor (Ratnam et al, 1999). A comparison of the crystal structures for the apo enzyme and the enzyme-NADPH binary complex suggests a conformational change takes place upon cofactor binding, similar to that observed in other AKRs (Cooper et al, 2007;Sanli and Blaber, 2001;Sanli et al, 2003). Using an arginine !…”

Human Cytosolic Hydroxysteroid Dehydrogenases of the Aldo-ketoreductase Superfamily Catalyze Reduction of Conjugated Steroids

“…Unfortunately, less is known about the mature structure of the ligand binding site of AKR1C4, because no ternary structure is available. Because it is known that the binding sites of AKR1C enzymes undergo significant conformational change when the binary complex is converted to the ternary complex (18,19), docking of the steroid conjugates into AKR1C4 was not further pursued.…”