Single-molecule enzymology of steroid transforming enzymes: Transient kinetic studies and what they tell us

Abstract

Structure-function studies on steroid transforming enzymes often use site-directed mutagenesis to inform mechanisms of catalysis and effects on steroid binding, and data are reported in terms of changes in steady state kinetic parameters kcat, Km and kcat/Km. However, this dissection of function is limited since kcat is governed by the rate-determining step and Km is a complex macroscopic kinetic constant. Often site-directed mutagenesis can lead to a change in the rate-determining step which cannot be revealed by just reporting a decrease in kcat alone. These issues are made more complex when it is considered that many steroid transforming enzymes have more than one substrate and product. We present the case for using transient-kinetics performed with stopped-flow spectrometry to assign rate constants to discrete steps in these multi-substrate reactions and their use to interpret enzyme mechanism and the effects of disease and engineered mutations. We demonstrate that fluorescence kinetic transients can be used to measure ligand binding that may be accompanied by isomerization steps, revealing the existence of new enzyme intermediates. We also demonstrate that single-turnover reactions can provide a klim for the chemical step and Ks for steroid-substrate binding and that when coupled with kinetic isotope effect measurements can provide information on transition state intermediates. We also demonstrate how multiple turnover experiments can provide evidence for either "burst-phase" kinetics, which can reveal a slow product release step, or linear-phase kinetics, in which the chemical step can be rate-determining. With these assignments it becomes more straightforward to analyze the effects of mutations. We use examples from the hydroxysteroid dehydrogenases (AKR1Cs) and human steroid 5β-reductase (AKR1D1) to illustrate the utility of the approach, which are members of the aldo-keto reductase (AKR) superfamily.

Keywords: Aldo-keto reductase; Hydroxysteroid dehydrogenase; Kinetic-isotope effect; Steroid double-bond reductases; Stopped-flow; Transient-kientics.

Fig. 1

Stopped-flow instrument. (A) Schematic of stopped-flow principle; (B) Components of Applied Photophysics stopped-flow instrument.

Fig. 2

Ordered Bi–Bi mechanism for aldo-keto reductases. Schematic of ordered bi–bi mechanism for AKR1C enzymes catalyzing the reduction of 5α-DHT using the Cleland notation. E = enzyme; S = steroid substrate; and P = steroid product. 3α-diol, 5α-androstan-3α,17β-diol.

Fig. 3

Isomerization events associated with NADP(H) binding to AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Global simulation of progress curves for the binding of NADPH to AKR1C2. Experimental (gray) and simulated (black) progress curves of protein fluorescence at 330 nm are shown. The samples contained 0.5 μM AKR1C2 and 5.0, 7.5, and 12.5 μM NADPH (top to bottom in panel A) (all final concentrations). (B) Secondary plots of the observed rate constants vs [NADPH] fitted to a hyperbolic function using multiple concentrations of NADPH. Adapted from reference .

Fig. 4

Ligand chase experiments from AKR1D1 and the P133R mutant. NADP⁺ is chased from the E·NADP⁺ complex by NADPH. The P133R mutant (red) showed a much faster rate of releae of NADP⁺ than the WT (green). An enzyme-NADP⁺ solution (AKR1D1, 1 μM; NADP⁺, 5 μM) from one syringe was rapidly mixed (1:1) with a NADPH solution (100 μM) from the second syringe in the stopped-flow. In the control experiments, the enzyme solution or the buffer were mixed with the NADPH solution in the stopped-flow (blue). Experiments were performed in 100 mM potassium phosphate pH 7.0. Reproduced with permission from the American Chemical Society, Biochemistry (2015) [Epub ahead of print Sep 29]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Transition-state intermediates for hydroxysteroid dehydrogenases. Three possible mechanisms are shown with different consequences for KIEs. When hydride transfer occurs first an oxyanion intermediate is expected and will yield a solvent KIE > primary KIE. When protonation occurs first a carbocation intermediate is expected and a primary KIE > solvent KIE. When a concerted mechanism occurs a neutral intermediate is expected and the primary and solvent KIE will be similar.

Fig. 6

Single turnover experiments for the reduction of DHT by AKR1C2. Global simulation of progress curves for NADPH dependent (A) and NADPD-dependent (B) single turnover of DHT catalyzed by AKR1C2. Experimental (gray) and simulated (black) progress curves of the decrease in NADPH fluorescence at 450 nm are shown. The samples contained 0.45 μM AKR1C2, 0.4 μM NADPH or NADPD, and 5, 15, and 20 μM DHT (traces from top to bottom). For clarity, data for other concentrations of DHT are not shown but were used in the global fitting. (C) k_obs for the individual progress curves observed with increasing DHT using fixed concentrations of NADPH or NADPD were obtained and the hyperbolic equation fit to the k_obs. Adapted from reference .

Fig. 7

Discrimination of fast and slow substrates for AKR1D1 under multiple turnover conditions. For stopped-flow multiple turnover experiments, the enzyme–cofactor solution contained a fixed concentration of AKR1D1 and excess NADPH, and was mixed with excess steroid substrate. A typical reaction contained 2.5 μM AKR1D1, 18 μM NADPH and 25 μM steroid. Either the burst equation (Eq. (4)) or the linear equations (Eq. (3)) were fit to averaged reaction traces from at least three replicates. (A) Averaged progress curves of decreases in NADPH fluorescence for samples containing testosterone (○), cortisone (▽), and aldosterone (△); (B) averaged progress curves of decreases in NADPH fluorescence for samples containing no steroid (⋄), cholestenone (+), or 7α-hydroxycholest-4-en-3-one (×). (C) fitting of the burst-equation to an averaged trace of a cortisone multiple turnover reaction; (D) fitting of the linear equation to an averaged trace of a 7α-hydroxycholest-4-en-3-one multiple turnover reaction. The initial increase in fluorescence signal in the reaction of 7α-hydroxycholest-4-en-3-one was also observed in the sample containing no steroid and was not used in data fitting. Fitted lines are in grey and are mostly superimposed by the actual data points. Residual plots demonstrate the quality of the fit. Reproduced with permission from Portland Press. This Figure was originally published by Y. Jin, M. Chen, and T.M. Penning in the Biochem. J. 462 (2014) 163–171.