Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice.

There is an increasing trend in studies utilizing cell-specific deletion of genes through conditional gene deletion by CRE recombination. Despite numerous advantages, this strategy also has limitations such as ectopic CRE-expression and germline recombination. Two commonly used gonadotropin-releasing hormone (Gnrh)-driven CRE-expressing mice both target GnRH neurons. However, a direct comparison of the cells targeted and their phenotypic outcome have not yet been presented. To compare where recombination takes place, we crossed the Gnrh-cre and Lhrh-cre lines with the Rosa26-LacZ reporter mouse. Lhrh-cre allowed recombination of the Rosa26-LacZ gene in ∼700 cells, which is comparable to the GnRH neuronal population. Surprisingly, there were > 20 times more LacZ expressing cells in the adult Gnrh-cre:Rosa26-LacZ than the Lhrh-cre:Rosa26-LacZ brain. The greatest differences in targeting of the Gnrh-cre and Lhrh-cre lines were found in the septum, the suprachiasmatic nucleus, and the septohypothalamic area. This difference in cells targeted was present from embryonic day 12. A prior study using the Gnrh-cre to delete the transcription factor Otx2 found fewer GnRH neurons, leading to male and female subfertility. To recapitulate this study, we performed a fertility assay in Otx2:Lhrh-cre mice. We confirmed the requirement for Otx2 in GnRH neuron development, fertility and correct gonadotropin hormone release in Otx2:Lhrh-cre males, but the subfertility was more modest than in Otx2:Gnrh-cre and absent in female Otx2:Lhrh-cre. This suggests that ectopic expression of Gnrh-cre contributes to the reproductive phenotype observed. Finally, the Cre alleles caused germline recombination of the flox allele when transmitted from either parent, generating embryonic lethal knock-out offspring, producing smaller live litters.

Characterization of the recombinant (R)- and (S)-hydroxypropyl-coenzyme M dehydrogenases: A case study to augment the teaching of enzyme kinetics and stereoselectivity.

A homologous pair of stereospecific NAD-dependent enzymes, (R)- and (S)-hydroxypropyl-coenzyme M dehydrogenase, are part of a bacterial pathway of short-chain alkene and epoxide metabolism. Their discovery and study, which spans multiple publications over more than a decade, is a data rich story that combines both classical and contemporary experimental biochemistry. A subset of the data for characterization of the recombinant enzymes was used as a case study to augment the teaching of enzyme kinetics and stereoselectivity in an undergraduate biochemistry course at California State University-Chico. © 2018 International Union of Biochemistry and Molecular Biology, 47(2): 124-132, 2019.

Preliminary investigation of deoxyoligonucleotide binding to ribonuclease A using mass spectrometry: An attempt to develop a lab experience for undergraduates.

Deoxyoligonucleotide binding to bovine pancreatic ribonuclease A (RNase A) was investigated using electrospray ionization ion-trap mass spectrometry (ESI-IT-MS). Deoxyoligonucleotides included CCCCC (dC 5) and CCACC (dC 2AC 2).  This work was an attempt to develop a biochemistry lab experience that would introduce undergraduates to the use of mass spectrometry for the analysis of protein-ligand interactions.  Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations.  Samples at equilibrium were infused directly into the mass spectrometer under native conditions.  For each deoxyoligonucleotide, mass spectra showed one-to-one binding stoichiometry, with marked increases in the total ion abundance of ligand-bound RNase A complexes as a function of concentration, but the accurate determination of dC 5 and dC 2AC 2 dissociation constants was problematic.

Virtual protein purification: A simple exercise to introduce ph as a parameter that effects ion exchange chromatography.

This article describes a simple exercise using a free, easy-to-use, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to pH as a parameter that affects anion-exchange chromatography. The exercise was tested with biochemistry majors at California State University-Chico. Given the versatility of the program, this work is also a model for instructors that wish to develop their own exercise to help teach other protein purification techniques. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):91-97, 2018.

Bmal1 Is Required for Normal Reproductive Behaviors in Male Mice.

Circadian rhythms synchronize physiological processes with the light-dark cycle and are regulated by a hierarchical system initiated in the suprachiasmatic nucleus, a hypothalamic region that receives direct photic input. The suprachiasmatic nucleus then entrains additional oscillators in the periphery. Circadian rhythms are maintained by a molecular transcriptional feedback loop, of which brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a key member. Disruption of circadian rhythms by deletion of the BMAL1 gene (Bmal1 knockout [KO]) induces a variety of disease states, including infertility in males, due to unidentified mechanisms. We find that, despite normal sperm function, Bmal1 KO males fail to mate with receptive females, indicating a behavioral defect. Mating is dependent on pheromone detection, as are several other behaviors. We determined that Bmal1 KO males also fail to display aggression and avoidance of predator scent, despite intact main olfactory function. Moreover, the vomeronasal organ, a specialized pheromone-responsive organ, was also functionally intact, as determined by calcium imaging in response to urine pheromone stimulus. However, neural circuit tracing using c-FOS activation revealed that, although Bmal1 KO males displayed appropriate activation in the olfactory bulb and accessory olfactory bulb, the bed nucleus of the stria terminalis and the medial preoptic area (areas responsible for integration of copulatory behaviors) failed to activate highly in response to the female scent. This indicates that neural signaling in select behavioral centers is impaired in the absence of BMAL1, likely underlying Bmal1 KO male copulatory defects, demonstrating the importance of the BMAL1 protein in the maintenance of neural circuits that drive pheromone-mediated mating behaviors.

Gene dosage of Otx2 is important for fertility in male mice.

Together, the hypothalamus, pituitary and gonads direct the development and regulation of reproductive function in mammals. Gonadotropin-releasing hormone (GnRH) expression is limited to ∼800 neurons that originate in the olfactory placode then migrate to the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal (HPG) axis is dependent upon correct neuronal migration of GnRH neurons into the hypothalamus followed by proper synthesis and pulsatile secretion of GnRH. Defects in any one of these processes causes infertility. Otx2, the vertebrate homologue of Drosophila orthodenticle, is a transcription factor that has been shown to be critical for normal brain and eye development and is expressed in both the developing GnRH neurons and the pituitary, suggesting that this gene may play a critical role in development of the HPG axis. As Otx2-null mice are embryonic lethal, we have analyzed the reproductive capacity of heterozygous Otx2 mice to determine the contribution of Otx2 gene dosage to normal HPG axis function. Our data reveal that correct dosage of Otx2 is critical for normal fertility as loss of one allele of Otx2 leads to a discernible reproductive phenotype in male mice due to disruption of the migration of GnRH neurons during development.

Aberrant development of the suprachiasmatic nucleus and circadian rhythms in mice lacking the homeodomain protein Six6.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is the central pacemaker for peripheral and organismal circadian rhythms. The development of this hypothalamic structure depends on genetic programs throughout embryogenesis. We have investigated the role of the homeodomain transcription factor Six6 in the development of the SCN. We first showed that Six6 mRNA has circadian regulation in the mouse SCN. We then characterized the behavioral activity patterns of Six6-null mice under various photoperiod manipulations and stained their hypothalami using SCN-specific markers. Six6-null mice display abnormal patterns of circadian behavior indicative of SCN abnormalities. The ability of light exposure to reset rhythms correlates with the presence or absence of optic nerves, but all Six6-null mice show irregular rhythms. In contrast, wild-type mice with crushed optic nerves maintain regular rhythms regardless of light exposure. Using immunohistochemistry for arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), and ß-galactosidase, we demonstrated the lack of these SCN markers in all Six6-null mice regardless of the presence of optic nerve or partial circadian rhythms. Therefore, Six6 is required for the normal development of the SCN, and the Six6-null mouse can mount independent, although irregular, circadian rhythms despite the apparent absence of a histochemically defined SCN.

Hypothalamic dysregulation and infertility in mice lacking the homeodomain protein Six6.

The hypothalamus, pituitary, and gonads coordinate to direct the development and regulation of reproductive function in mammals. Control of the hypothalamic-pituitary-gonadal axis is dependent on correct migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus, followed by proper synthesis and pulsatile secretion of GnRH, functions absent in patients with hypogonadal hypogonadism. In this study, we identify sine oculis-related homeobox 6 (Six6) as a novel factor necessary for proper targeting of GnRH expression to the limited population of GnRH neurons within the adult mouse hypothalamus and demonstrate that it is required for proper reproductive function in both male and female mice. Female Six6-null mice exhibit a striking decrease in fertility, failing to progress through the estrous cycle normally, show any signs of successful ovulation, or produce litters. Although basal gonadotropin production in these mice is relatively normal, analysis of GnRH expression reveals a dramatic decrease in total GnRH neuron numbers. We show that expression of Six6 is dramatically increased during GnRH neuronal maturation and that overexpression of Six6 induces GnRH transcription in neuronal cells. Finally, we demonstrate that this induction in GnRH expression is mediated via binding of Six6 to evolutionarily conserved ATTA sites located within the GnRH proximal promoter. Together, these data indicate that Six6 plays an important role in the regulation of GnRH expression and hypothalamic control of fertility.

Shotgun proteomics of Xanthobacter autotrophicus Py2 reveals proteins specific to growth on propylene.

Coenzyme M (CoM, 2-mercaptoethanesulfonate), once thought to be exclusively produced by methanogens, is now known to be the central cofactor in the metabolism of short-chain alkenes by a variety of aerobic bacteria. There is little evidence to suggest how, and under what conditions, CoM is biosynthesized by these organisms. A shotgun proteomics approach was used to investigate CoM-dependent propylene metabolism in the Gram-negative bacterium Xanthobacter autotrophicus Py2. Cells were grown on either glucose or propylene, and the soluble proteomes were analyzed. An average of 395 proteins was identified from glucose-grown replicates, with an average of 419 identified from propylene-grown replicates. A number of linear megaplasmid (pXAUT01)-encoded proteins were found to be specifically produced by growth on propylene. These included all known to be crucial to propylene metabolism, in addition to an aldehyde dehydrogenase, a DNA-binding protein, and five putative CoM biosynthetic enzymes. This work has provided fresh insight into bacterial alkene metabolism and has generated new targets for future studies in X. autotrophicus Py2 and related CoM-dependent alkene-oxidizing bacteria.

Mechanism of inhibition of aliphatic epoxide carboxylation by the coenzyme M analog 2-bromoethanesulfonate.

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys(82)) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys(87) was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys(82). These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.

Structural basis for stereoselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases.

Epoxide metabolism in Xanthobacter autotrophicus Py2 results in the conversion of epoxypropane to acetoacetate. Epoxide metabolism is initiated by the nucleophilic addition of coenzyme M to the (R)- and (S)-enantiomers of epoxypropane which forms the respective enantiomers of 2-hydroxypropyl-coenyme M. The (R)- and (S)-enantiomers of 2-hydroxypropyl coenzyme are oxidized to the achiral product 2-ketopropyl-CoM by two stereoselective dehydrogenases. The dehydrogenases catalyzing these reactions, termed (R)-hydroxypropyl-coenzyme M dehydrogenase (R-HPCDH) and (S)-hydroxypropyl-coenzyme M dehydrogenase (S-HPCDH), are NAD(+)-dependent enzymes belonging to the short chain dehydrogenase/reductase (SDR) family of enzymes. In this study, the crystal structure of R-HPCDH cocrystallized in the presence of (S)-hydroxypropyl-coenzyme M has been determined using X-ray diffraction methods and refined to 1.8 A resolution. The structure of R-HPCDH is tetrameric and stabilized by the interaction of the terminal carboxylates of each subunit with divalent metal ions. The structure of the presumed product-bound state reveals that binding interactions between the negatively charged oxygen atoms of the sulfonate moiety have striking similarities to sulfonate interactions observed in the previously determined structure of 2-ketopropyl-CoM oxidoreductase/carboxylase, highlighting the utility of coenzyme M as a carrier molecule in the pathway. The key elements of the aforementioned interactions are electrostatic interactions between the sulfonate oxygen atoms and two arginine residues (R152 and R196) of R-HPCDH. The comparison of the structure of R-HPCDH with a homology model of S-HPCDH provides a structural basis for a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for hydride abstraction.

Mechanistic implications of the structure of the mixed-disulfide intermediate of the disulfide oxidoreductase, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

The structure of the mixed, enzyme-cofactor disulfide intermediate of ketopropyl-coenzyme M oxidoreductase/carboxylase has been determined by X-ray diffraction methods. Ketopropyl-coenzyme M oxidoreductase/carboxylase belongs to a family of pyridine nucleotide-containing flavin-dependent disulfide oxidoreductases, which couple the transfer of hydride derived from the NADPH to the reduction of protein cysteine disulfide. Ketopropyl-coenzyme M oxidoreductase/carboxylase, a unique member of this enzyme class, catalyzes thioether bond cleavage of the substrate, 2-ketopropyl-coenzyme M, and carboxylation of what is thought to be an enzyme-stabilized enolacetone intermediate. The mixed disulfide of 2-ketopropyl-coenzyme M oxidoreductase/carboxylase was captured through crystallization of the enzyme with the physiological products of the reaction, acetoacetate, coenzyme M, and NADP, and reduction of the crystals with dithiothreitol just prior to data collection. Density in the active-site environment consistent with acetone, the product of reductive decarboxylation of acetoacetate, was revealed in this structure in addition to a well-defined hydrophobic pocket or channel that could be involved in the access for carbon dioxide. The analysis of this structure and that of a coenzyme-M-bound form provides insights into the stabilization of intermediates, substrate carboxylation, and product release.

Fluorescence-based cloning of a protein tyrosine kinase with a yeast tribrid system.

Post-translational modifications of proteins control myriad biological functions. However, relatively few methods exist for the identification of the enzymes that catalyze these modifications. To expand this repertoire, we report a yeast genetic approach that enables the identification of protein tyrosine kinases (PTKs) from cDNA libraries. Yeasts were transformed with four vectors encoding: 1) a potentially universal PTK substrate fused to the LexA DNA binding domain, 2) the Grb2-SH2 domain fused to the B42 activation domain, 3) a fluorescent reporter gene controlled by LexA DNA sites, and 4) a Jurkat cDNA library. Transient expression of PTKs, such as the lymphocyte-specific kinase Fyn, resulted in phosphorylation of the DNA-bound substrate, recruitment of the Grb2-SH2 domain, and activation of the fluorescent reporter gene. This brief induction of protein expression circumvented the potential toxicity of PTKs to the yeast. Fluorescence activated cell sorting (FACS) enabled isolation of PTKs, and these enzymes were further characterized by flow cytometry and immunoblotting. This approach provides a potentially general method for the identification and evaluation of enzymes involved in the post-translational modification of proteins.

The stereoselectivity and catalytic properties of Xanthobacter autotrophicus 2-[(R)-2-Hydroxypropylthio]ethanesulfonate dehydrogenase are controlled by interactions between C-terminal arginine residues and the sulfonate of coenzyme M.

2-[(R)-2-Hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase (DH) catalyzes the reversible oxidation of R-HPC to 2-(2-ketopropylthio)ethanesulfonate (2-KPC) in a key reaction in the bacterial conversion of chiral epoxides to beta-keto acids. R-HPCDH is highly specific for the R-enantiomer of HPC, while a separate enzyme, S-HPCDH, catalyzes the oxidation of the corresponding S-enantiomer. In the present study, the features of substrate and enzyme imparting stereospecificity have been investigated for R-HPCDH. S-HPC was a substrate for R-HPCDH with a K(m) identical to that for R-HPC but with a k(cat) 600 times lower. Achiral 2-propanol and short-chain (R)- and (S)-2-alkanols were substrates for R-HPCDH. For (R)-alkanols, as the carbon chain length increased, K(m) decreased, with the K(m) for (R)-2-octanol being 1700 times lower than for 2-propanol. At the same time, k(cat) changed very little and was at least 90% lower than k(cat) for R-HPC and at least 22 times higher than k(cat) for S-HPC. (S)-2-Butanol and (S)-2-pentanol were substrates for R-HPCDH. The K(m) for (S)-2-butanol was identical to that for (R)-2-butanol, while the K(m) for (S)-2-pentanol was 7.5 times higher than for (R)-2-pentanol. Longer chain (S)-2-alkanols were sufficiently poor substrates for R-HPCDH that kinetic parameters could not be determined. Mutagenesis of C-terminal arginine residues of R-HPCDH revealed that R152 and R196 are essential for effective catalysis with the natural substrates R-HPC and 2-KPC but not for catalysis with 2-alkanols or ketones as substrates. Short-chain alkylsulfonates and coenzyme M (2-mercaptoethanesulfonate) were found to modify the kinetic parameters for 2-butanone reduction by R-HPCDH in a saturable fashion, with the general effect of increasing k(cat), decreasing K(m), and increasing the enantioselectivity of 2-butanone reduction to a theoretical value of 100% (S)-2-butanol. The modulating effects of ethanesulfonate and propanesulfonate provided thermodynamic binding constants close to K(m) for the natural substrates R-HPC and 2-KPC. The effects of alkylsulfonates on modulating the enantioselectivity and kinetic properties of R-HPCDH were abolished in R152A and R196A mutants but not in mutants of other C-terminal arginine residues. Collectively, the results suggest that interactions between the sulfonate of CoM and specific arginine residues are key to the enantioselectivity and catalytic efficiency of R-HPCDH. A model is proposed wherein sulfonate-arginine interactions within an alkylsulfonate binding pocket control the catalytic properties of R-HPCDH.

Analysis of protein tyrosine kinase inhibitors in recombinant yeast lacking the ERG6 gene.

Studies of small-molecule-protein interactions in yeast can be hindered by the limited permeability of yeast to small molecules. This diminished permeability is thought to be related to the unique sterol composition of fungal membranes, which are enriched in the steroid ergosterol. We report the construction of the novel Saccharomyces cerevisiae yeast strain DCY250, which is compatible with yeast two-hybrid-based systems and bears a targeted disruption of the ERG6 gene to ablate ergosterol biosynthesis and enhance permeability to small molecules. The small-molecule inhibitors of protein tyrosine kinases (PTKs) PP1, PP2, herbimycin A, and staurosporine were investigated with yeast tribrid systems that detect the activity of the PTKs v-Abl and v-Src. These tribrid systems function by expression of the PTK, a B42 activation domain fused to the phosphotyrosine-binding Grb2 SH2 domain, a DNA-bound LexA-GFP-(AAYANAA)(4) universal PTK substrate, and a lacZ reporter gene. Yeast genetic systems that lack functional ERG6 were found to be as much as 20-fold more sensitive to small-molecule inhibitors of PTKs than systems with ERG6, and these deficient systems may provide a useful platform for the discovery and analysis of small-molecule-protein interactions.

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).

Crystallization and preliminary X-ray analysis of an R-2-hydroxypropyl-coenzyme M dehydrogenase.

The R-2-hydroxypropyl-coenzyme M (2-mercaptoethanesulfonate) dehydrogenase is a key enzyme in the microbial conversion of propylene to the central metabolite acetoacetate. This enzyme is an interesting member of the NAD(P)H-dependent short-chain dehydrogenase/reductase (SDR) family of enzymes, being one of a pair of homologous dehydrogenases that act in concert in a single pathway to convert the R- and S-enantiomers of hydroxypropyl-coenzyme M to the achiral ketopropyl-coenzyme M product. Crystallization trials have revealed that the highest diffraction quality crystals (better than 2.0 A resolution) could be achieved when the reaction substrates were added to the enzyme in a stoichiometric excess prior to crystallization.

Characterization of the 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase from Xanthobacter strain Py2: product inhibition, pH dependence of kinetic parameters, site-directed mutagenesis, rapid equilibrium inhibition, and chemical modification.

Although the short-chain dehydrogenase/reductase (SDR) superfamily contains a very large number of members defined in annotated databases and by biochemical and structural studies, very few SDR enzymes have been identified that have a homologous partner catalyzing the same reaction but with an opposite stereospecificity. In the present study we have cloned and expressed one of these enzymes, the 2-[(R)-2-hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase, that is part of the coenzyme M-dependent pathway of alkene and epoxide metabolism in Xanthobacter strain Py2. Investigation of the kinetic mechanism using product inhibition suggested that a compulsory-ordered ternary complex mechanism was followed. The pH dependence of k(cat)/K(m) indicated the presence of a single ionizable residue of catalytic importance (pK(a) = 6.9) that was proposed to be Y155 of the catalytic triad. Amino acid substitutions of the putative catalytic triad residues produced inactive enzymes (S142C, Y155F, Y155E, and K159A) or enzyme with a greatly decreased activity (S142A). Inhibitors were investigated as probes of the molecular features of R-HPC that contribute to substrate binding. 2-[(S)-2-Hydroxypropylthio]ethanesulfonate (S-HPC) and 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate were found to be competitive inhibitors of R-HPC with K(ic) values close to the K(m) for R-HPC. The arginine-specific modifiers 2,3-butanedione and phenylglyoxal were found to be inactivators, and inactivation could be protected against by the addition of R-HPC. 2,3-Butanedione was found to reduce enzyme activity with R-HPC as a substrate much more dramatically than with substrates that lacked a sulfonate moiety [e.g., 2-propanol, (R)-2-pentanol, and (R)-2-heptanol]. Amino acid analyses of enzyme modified by 2,3-butanedione in the presence and absence of S-HPC suggested protection of a single arginine residue. On the basis of these results, we propose that one or more active site arginines play a key role in substrate binding via an ionic interaction with the sulfonate moiety of R-HPC.

Rapid detection of protein tyrosine kinase activity in recombinant yeast expressing a universal substrate.

Yeast two-hybrid systems are powerful proteomics tools for the discovery of protein-protein interactions. However, these systems are typically unable to detect interactions dependent on post-translational modifications such as tyrosine phosphorylation. We report a novel yeast tribrid system that expresses a potentially universal protein tyrosine kinase (PTK) substrate to detect diverse PTKs. Validation with the oncogenic kinases v-Abl and v-Src, which exhibit divergent substrate specificities, demonstrated significant potential for cloning PTKs en masse from cDNA libraries.