Characterization of human adrenal cytochrome P450 11B2 products of progesterone and androstenedione oxidation.

Cytochrome P450 (P450) 11B1 and 11B2 both catalyze the 11ß-hydroxylation of 11-deoxycorticosterone and the subsequent 18-hydroxylation of the product. P450 11B2, but not P450 11B1, catalyzes a further C-18 oxidation to yield aldosterone. 11-Oxygenated androgens are of interest, and 11-hydroxy progesterone has been reported to be a precursor of these. Oxidation of progesterone by purified recombinant P450 11B2 yielded a mono-hydroxy derivative as the major product, and co-chromatography with commercial standards and 2-D NMR spectroscopy indicated 11ß-hydroxylation. 18-Hydroxyprogesterone and a dihydroxyprogesterone were also formed. Similarly, oxidation of androstenedione by P450 11B2 yielded 11ß-hydroxyandrostenedione, 18-hydroxyandrostenedione, and a dihydroxyandrostenedione. The steady-state kinetic parameters for androstenedione and progesterone 11ß-hydroxylation were similar to those reported for the classic substrate 11-deoxycorticosterone. The source of 11α-hydroxyprogesterone in humans remains unresolved.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes.

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6-200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 µM Adx, and higher concentrations were not inhibitory up to at least 50 µM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

Human cytochrome P450 11B2 produces aldosterone by a processive mechanism due to the lactol form of the intermediate 18-hydroxycorticosterone.

Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme (i.e. processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.

Conformational selection dominates binding of steroids to human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase.

Enzymes are known to exhibit conformational flexibility. An important consequence of this flexibility is that the same enzyme reaction can occur via multiple reaction pathways on a reaction landscape. A model enzyme for the study of reaction landscapes is lactate dehydrogenase. We have previously used temperature-jump (T-jump) methods to demonstrate that the reaction landscape of lactate dehydrogenase branches at multiple points creating pathways with varied reactivity. A limitation of this previous work is that the T-jump method makes only small perturbations to equilibrium and may not report conclusively on all steps in a reaction. Therefore, interpreting T-jump results of lactate dehydrogenase kinetics has required extensive computational modeling work. Rapid mixing methods offer a complementary approach that can access large perturbations from equilibrium; however, traditional enzyme mixing methods like stopped-flow do not allow for the observation of fast protein dynamics. In this report, we apply a microfluidic rapid mixing device with a mixing time of <100 µs that allows us to study these fast dynamics and the catalytic redox step of the enzyme reaction. Additionally, we report UV absorbance and emission T-jump results with improved signal-to-noise ratio at fast times. The combination of mixing and T-jump results yields an unprecedented view of lactate dehydrogenase enzymology, confirming the timescale of substrate-induced conformational change and presence of multiple reaction pathways.

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.

Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of <0.2mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.

Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase.

Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein-ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.

Sandwich-format 3D printed microfluidic mixers: a flexible platform for multi-probe analysis.

We report on a microfluidic mixer fabrication platform that increases the versatility and flexibility of mixers for biomolecular applications. A sandwich-format design allows the application of multiple spectroscopic probes to the same mixer. A polymer spacer is 'sandwiched' between two transparent windows, creating a closed microfluidic system. The channels of the mixer are defined by regions in the polymer spacer that lack material and therefore the polymer need not be transparent in the spectral region of interest. Suitable window materials such as CaF2 make the device accessible to a wide range of optical probe wavelengths, from the deep UV to the mid-IR. In this study, we use a commercially available 3D printer to print the polymer spacers to apply three different channel designs into the passive, continuous-flow mixer, and integrated them with three different spectroscopic probes. All three spectroscopic probes are applicable to each mixer without further changes. The sandwich-format mixer coupled with cost-effective 3D printed fabrication techniques could increase the applicability and accessibility of microfluidic mixing to intricate kinetic schemes and monitoring chemical synthesis in cases where only one probe technique proves insufficient.

Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy.

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm(-1), respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions.

Submillisecond mixing in a continuous-flow, microfluidic mixer utilizing mid-infrared hyperspectral imaging detection.

We report a continuous-flow, microfluidic mixer utilizing mid-infrared hyperspectral imaging detection, with an experimentally determined, submillisecond mixing time. The simple and robust mixer design has the microfluidic channels cut through a polymer spacer that is sandwiched between two IR transparent windows. The mixer hydrodynamically focuses the sample stream with two side flow channels, squeezing it into a thin jet and initiating mixing through diffusion and advection. The detection system generates a mid-infrared hyperspectral absorbance image of the microfluidic sample stream. Calibration of the hyperspectral image yields the mid-IR absorbance spectrum of the sample versus time. A mixing time of 269 µs was measured for a pD jump from 3.2 to above 4.5 in a D2O sample solution of adenosine monophosphate (AMP), which acts as an infrared pD indicator. The mixer was further characterized by comparing experimental results with a simulation of the mixing of an H2O sample stream with a D2O sheath flow, showing good agreement between the two. The IR microfluidic mixer eliminates the need for fluorescence labeling of proteins with bulky, interfering dyes, because it uses the intrinsic IR absorbance of the molecules of interest, and the structural specificity of IR spectroscopy to follow specific chemical changes such as the protonation state of AMP.

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics.

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 µs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 µs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 µs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

Sequence length dictates repeated CAG folding in three-way junctions.

The etiology of a large class of inherited neurological diseases is founded on hairpin structures adopted by repeated DNA sequences, and this folding is determined by base sequence and DNA context. Using single substitutions of adenine with 2-aminopurine, we show that intrastrand folding in repeated CAG trinucleotides is also determined by the number of repeats. This isomeric analogue has a fluorescence quantum yield that varies strongly with solvent exposure, thereby distinguishing particular DNA motifs. Prior studies demonstrated that (CAG)(8) alone favors a stem-loop hairpin, yet the same sequence adopts an open loop conformation in a three-way junction. This comparison suggests that repeat folding is disrupted by base pairing in the duplex arms and by purine-purine mismatches in the repeat stem. However, these perturbations are overcome in longer CAG repeats, as demonstrated by studies of isolated and integrated forms of (CAG)(15). The oligonucleotide alone forms a symmetrically folded hairpin with looplike properties exhibited by the relatively high emission intensities from a modification in the central eighth repeat and with stemlike properties evident from the relatively low emission intensities from peripheral modifications. Significantly, these hairpin properties are retained when (CAG)(15) is integrated into a duplex. Intrastrand folding by (CAG)(15) in the three-way junction contrasts with the open loop adopted by (CAG)(8) in the analogous context. This distinction suggests that cooperative interactions in longer repeat tracts overwhelm perturbations to reassert the natural folding propensity. Given that anomalously long repeats are the genetic basis of a large class of inherited neurological diseases, studies with (CAG)-based three-way junctions suggest that their secondary structure is a key factor in the length-dependent manifestation and progression of such diseases.

Structural studies of a trinucleotide repeat sequence using 2-aminopurine.

The secondary structure of repeated trinucleotide sequences results in the development of several neurodegenerative diseases, and these studies consider the (CAG)(8) sequence that forms a stem-loop hairpin. The structural and thermodynamic properties of this hairpin are assessed using 2-aminopurine substitutions for adenine at six positions in this repeated sequence. Circular dichroism spectra and thermal denaturation experiments show that the secondary structure is not disturbed by the modifications. The local structure of the hairpin was monitored using the fluorescence intensities of 2-aminopurines, the changes in the intensity relative to the denatured state, and the sensitivity of the fluorescence to quenching by acrylamide. To establish the stem and loop characteristics in (CAG)(8), known reference points for stem, loop, and exposed base motifs were used. In the vicinity of the loop, the bases become more solvent exposed, which suggests that the instability associated with this repeated hairpin influences the global secondary structure. These results provide the basis to interpret the structures adopted by other repeated (CAG) structures.