H-dependent Oxidoreductases Research Articles

Hunting for low abundant redox proteins in plant plasma membranes

Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction

The role of NAD(P)H-dependent oxidoreductases of the outer mitochondrial membrane (OMM) in the activation of lipophilic cationic dyes is poorly understood. In the present study we compared the rates of production of reactive oxygen species (ROS) and mitochondriotoxic effects of the redox-cycling lipophilic cationic dye lucigenin upon its activation by the respiratory chain and NAD(P)H-dependent oxidoreductases of the OMM. We found that, only in the presence of external NADH and NADPH, which are unable to penetrate the inner membrane, lucigenin stimulated a massive superoxide production and a fast permeabilization of mitochondrial membranes. The permeabilization was biphasic. The first, cyclosporin A-insensitive and Ca 2+-independent phase was characterized by increased permeability of the inner mitochondrial membrane to solutes with molecular masses of ≤200 Da. The second phase was sensitive to the antagonists of the permeability transition pore (mPTP) and was characterized by permeability similar to that of mPTP (≤1500 Da). A massive cytochrome c release was observed even at the first phase of permeability when the second phase was inhibited by mPTP antagonists. Whatever the site of lucigenin activation, antioxidants and scavengers of ROS that strongly decrease the ROS level were unable to delay or prevent the permeabilization of membranes, which casts doubt on the involvement of ROS in the regulation of permeability by redox-cycling lipophilic cations. Our results strongly support the idea that the NAD(P)H-dependent reductases of xenobiotics of the OMM can mediate the toxicity of cationic dyes.

Optimizing a Biocatalyst for Improved NAD(P)H Regeneration: Directed Evolution of Phosphite Dehydrogenase

Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by >6-fold. Large-scale production of the final mutant enzyme by fermentation resulted in approximately 6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.

Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelial-dependent vasodilatation.

The present study was aimed to evaluate the mechanisms involved in the vasorelaxant effects of red wine polyphenol compounds (RWPC) in small mesenteric rat arteries. RWPC produce relaxation in small mesenteric arteries. This relaxant effect was abolished by endothelial denudation, NO-synthase blockade with L-NAME and partial depolarization with KCl or L-NAME plus KCl. Incubation with the reactive oxygen species scavenger, superoxide dismutase (SOD) plus catalase, or inhibition of NAD(P)H-dependent oxidoreductases with diphenyleneiodonium also inhibited RWPC induced vascular relaxation. Application of RWPC elicited a transient increase in intracellular calcium concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC), which was attenuated by a mixture of SOD and catalase. Incubation of BAEC with RWPC increased the SOD inhibitable production of O2-. These results suggest the involvement of O2- in the [Ca2+]i increase evoked by RWPC, leading to the activation of enzymes involved in the release of endothelial relaxant factors and subsequent vasodilatation of resistance arteries.

Sphingolipid biosynthesis in pathogenic fungi: Identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus

An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p ( TSC10 ( YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.

Crystal Structures of Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Belonging to a New Family of NAD(P)H-dependent Oxidoreductases: CONFORMATIONAL CHANGE, SUBSTRATE RECOGNITION, AND STEREOCHEMISTRY OF THE REACTION

Delta(1)-Piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato belongs to a novel sub-class in a large family of NAD(P)H-dependent oxidoreductases distinct from the conventional MDH/LDH superfamily characterized by the Rossmann fold. We have determined the structures of the following three forms of the enzyme: the unliganded form, the complex with NADPH, and the complex with NADPH and pyrrole-2-carboxylate at 1.55-, 1.8-, and 1.7-A resolutions, respectively. The enzyme exists as a dimer, and the subunit consists of three domains; domain I, domain II (NADPH binding domain), and domain III. The core of the NADPH binding domain consists of a seven-stranded predominantly antiparallel beta-sheet fold (which we named SESAS) that is characteristic of the new oxidoreductase family. The enzyme preference for NADPH over NADH is explained by the cofactor binding site architecture. A comparison of the overall structures revealed that the mobile domains I and III change their conformations to produce the catalytic form. This conformational change plays important roles in substrate recognition and the catalytic process. The active site structure of the catalytic form made it possible to identify the catalytic Asp:Ser:His triad and investigate the catalytic mechanism from a stereochemical point of view.

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.

Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism.

Aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k (cat)/ K (m)=1100-10300 mM(-1).min(-1)), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K (i)=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from beta-carotene and the control of retinal bioavailability.

Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases: sequence-based classification and analysis of structure–function relationships

Multiple sequence alignment and analysis of evolutionary relationships have been used to characterize a family of polyol-specific long-chain dehydrogenases/reductases (PSLDRs). At the present time, 66 known and putative NAD(P)H-dependent oxidoreductases of mainly prokaryotic origin and between 357 and 544 amino acids in length constitute this family. The family is shown to include d-mannitol 2-dehydrogenase, d-mannonate 5-oxidoreductase, d-altronate 5-oxidoreductase, d-arabinitol 4-dehydrogenase, and d-mannitol-1-phosphate 5-dehydrogenase which form individual sub-families (defined by internal sequence identity of ⩾30%) having distant origin and divergent substrate specificity but clearly displaying entire-chain relationship. When all forms are aligned, only three residues, Gly-33, Asp-230, and Lys-295 (in the numbering of Pseudomonas fluorescens d-mannitol 2-dehydrogenase ( PsM2DH)) are strictly conserved. By combining sequence alignment with the known structure of PsM2DH and results from site-directed mutagenesis, we have developed a structure/function analysis for the family. Gly-33 is in the N-terminal coenzyme-binding domain and part of a nucleotide fingerprint region for the family, and Asp-230 and Lys-295 are at an interdomain segment contributing to the active site in which the lysine likely functions as the catalytic general acid/base. PSLDRs do not require a metal cofactor for activity and are specific for transferring the 4-pro- S hydrogen from NAD(P)H. Comparisons reveal that the core part of the two-domain fold has been conserved throughout all family members, perhaps reflecting the recruitment of a stable oxidoreductase structure and extensive trimming thereof to acquire functional properties specific to each sub-family. They also identify interactions that define the chemical mechanism of oxidoreduction and likely contribute to substrate and co-substrate specificities and are thus relevant for protein engineering.

Discussion

The diazaborine family of compounds have antibacterial properties against a range of gram-negative bacteria. Initially, this was thought to be due to the prevention of lipopolysaccharide synthesis. More recently, the molecular target of diazaborines has been identified as the NAD(P)H-dependent enoyl acyl carrier protein reductase (ENR), which catalyses the last reductive step of fatty acid synthase. ENR from Mycobacterium tuberculosis is the target for the front-line antituberculosis drug isoniazid. The emergence of isoniazid resistance strains of M. tuberculosis, a chronic infectious disease that already kills more people than any other infection, is currently causing great concern over the prospects for its future treatment, and it has reawakened interest in the mechanism of diazaborine action. Diazaborines only inhibit ENR in the presence of the nucleotide cofactor, and this has been explained through the analysis of the x-ray crystallographic structures of a number of Escherichia coli ENR-NAD+-diazaborine complexes that showed the formation of a covalent bond between the boron atom in the diazaborines and the 2'-hydroxyl of the nicotinamide ribose moiety that generates a noncovalently bound bisubstrate analogue. The similarities in catalytic chemistry and in the conformation of the nucleotide cofactor across the wider family of NAD(P)-dependent oxidoreductases suggest that there are generic opportunities to mimic the interactions seen here in the rational design of bisubstrate analogue inhibitors for other NAD(P)H-dependent oxidoreductases.

A hormogonium regulating locus, hrmUA, of the cyanobacterium Nostoc punctiforme strain ATCC 29133 and its response to an extract of a symbiotic plant partner Anthoceros punctatus.

Transposon-generated mutant strain UCD 328 of Nostoc punctiforme strain ATCC 29133 has a phenotype of an increased sensitivity to a hormogonium-inducing factor exuded by a symbiotic plant partner, Anthoceros punctatus, and an initial increased hormogonium-dependent infection of the plant. Sequence analysis showed that the transposition site in strain UCD 328 lies within a 1,251-bp open reading frame (ORF), designated hrmA, that displays no significant similarity to known database sequences. A second, 837-bp ORF (hrmU) ends 2 bp 5' from the start of hrmA and has the signature sequences belonging to a family of NAD(P)H-dependent oxidoreductases. Strains having insertional mutations in hrmU or hrmA reproduce the strain UCD 328 phenotype. Transcriptional fusions of luxAB to hrmU or hrmA show an 8- to 10-fold peak increase in luciferase activity 13 to 20 h after the start of incubation in the presence of an aqueous extract of A. punctatus. A promoter induced by the extract was deduced to be between 2.0 to 3.4 kb from the translational start of hrmU. A multicopy plasmid that contains hrmUA within a 6.2-kb fragment conferred an increased infection phenotype on wild-type N. punctiforme 29133. This plasmid and another plasmid containing 4.4 kb of DNA 5' of the transposition site prevented extract-dependent induction of hrmA-luxAB transcription in strain UCD 328, implicating titration of some trans-activator(s) by the cloned fragments. We hypothesize a role for hrmUA in the inhibition of hormogonium formation by the metabolism of an unknown hormogonium-regulating metabolite.

Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.

Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.

Transposon-generated mutant N10 of Anabaena sp. strain PCC 7120 has a Het- phenotype (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation reproduced a Het- phenotype, but reconstructions with other insertions at the position of the transposon produced strains that form multiple contiguous heterocysts. Sequence analysis around the site of insertion of the transposon showed that the insertion lies within the 5' end of an 861-bp open reading frame (ORF) (hetN). The product of translation of hetN (HetN) shows extensive similarity to NAD(P)H-dependent oxidoreductases that are involved in biosyntheses of fatty acids, poly-beta-hydroxybutyrate, nod factor, and polyketides. A second, 1,518-bp ORF (hetM) that ends 556 bp 5' from the start of hetN appears to encode a protein that has at least two functional domains: its amino terminus is similar to an acyl carrier protein, while its central portion is similar to domains of proteins that perform reductive reactions. A third, 711-bp ORF (hetI) encoded on the opposite strand ends 42 bp away from the 3' end of hetN. The protein encoded by hetI, HetI, is similar to Sfp from Bacillus subtilis and EntD from Escherichia coli, proteins that are required for the biosynthesis or export of cyclic peptides. Clones from a lambda-EMBL3 library that contain the wild-type DNA for hetN do not complement the hetN::Tn5-1063 mutation in N10. The presence of hetN, as the only ORF, on a replicating plasmid suppresses heterocyst formation in wild-type cells, whereas the additional presence of hetI alleviates this effect.

Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the 'fingerprint' region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles.

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindström, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of A novel A-ring pyrazole steroid

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17β-ol ( 3 ), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)β-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a K i of 90 ± 20 nM. The activities of 3α, 20β-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5α-reductase (from rat prostate), and 3α-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3 . Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.