Probing erectile function: S-(2-boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth muscle relaxation in human penile corpus cavernosum.

The boronic acid-based arginine analogue S-(2-boronoethyl)-L-cysteine (BEC) has been synthesized and assayed as a slow-binding competitive inhibitor of the binuclear manganese metalloenzyme arginase. Kinetic measurements indicate a K(I) value of 0.4-0.6 microM, which is in reasonable agreement with the dissociation constant of 2.22 microM measured by isothermal titration calorimetry. The X-ray crystal structure of the arginase-BEC complex has been determined at 2.3 A resolution from crystals perfectly twinned by hemihedry. The structure of the complex reveals that the boronic acid moiety undergoes nucleophilic attack by metal-bridging hydroxide ion to yield a tetrahedral boronate anion that bridges the binuclear manganese cluster, thereby mimicking the tetrahedral intermediate (and its flanking transition states) in the arginine hydrolysis reaction. Accordingly, the binding mode of BEC is consistent with the structure-based mechanism proposed for arginase as outlined in Cox et al. [Cox, J. D., Cama, E., Colleluori D. M., Pethe, S., Boucher, J. S., Mansuy, D., Ash, D. E., and Christianson, D. W. (2001) Biochemistry 40, 2689-2701.]. Since BEC does not inhibit nitric oxide synthase, BEC serves as a valuable reagent to probe the physiological relationship between arginase and nitric oxide (NO) synthase in regulating the NO-dependent smooth muscle relaxation in human penile corpus cavernosum tissue that is required for erection. Consequently, we demonstrate that arginase is present in human penile corpus cavernosum tissue, and that the arginase inhibitor BEC causes significant enhancement of NO-dependent smooth muscle relaxation in this tissue. Therefore, human penile arginase is a potential target for the treatment of sexual dysfunction in the male.

Biochemical and functional profile of a newly developed potent and isozyme-selective arginase inhibitor.

An increase in arginase activity has been associated with the pathophysiology of a number of conditions, including an impairment in nonadrenergic and noncholinergic (NANC) nerve-mediated relaxation of the gastrointestinal smooth muscle. An arginase inhibitor may rectify this condition. We compared the effects of a newly designed arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH), with the currently available N(omega)-hydroxy-L-arginine (L-HO-Arg), on the NANC nerve-mediated internal anal sphincter (IAS) smooth-muscle relaxation and the arginase activity in the IAS and other tissues. Arginase caused an attenuation of the IAS smooth-muscle relaxations by NANC nerve stimulation that was restored by the arginase inhibitors. L-HO-Arg but not ABH caused dose-dependent and complete reversal of N(omega)-nitro-L-arginine-suppressed IAS relaxation that was similar to that seen with L-arginine. Both ABH and L-HO-Arg caused an augmentation of NANC nerve-mediated relaxation of the IAS. In the IAS, ABH was found to be approximately 250 times more potent than L-HO-Arg in inhibiting the arginase activity. L-HO-Arg was found to be 10 to 18 times more potent in inhibiting the arginase activity in the liver than in nonhepatic tissues. We conclude that arginase plays a significant role in the regulation of nitric oxide synthase-mediated NANC relaxation in the IAS. The advent of new and selective arginase inhibitors may play a significant role in the discrimination of arginase isozymes and have important pathophysiological and therapeutic implications in gastrointestinal motility disorders.

Interaction of echicetin with a high affinity thrombin binding site on platelet glycoprotein GPIb.

Echicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin ( < 0.2 U/ml), by binding to platelet glycoprotein Ib (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin ( > 0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde.

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.

Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate.

Phosphoenolpyruvate carboxykinase from chicken liver mitochondria and rat liver cytosol catalyzes the phosphorylation of alpha-substituted carboxylic acids such as glycolate, thioglycolate, and DL-beta-chlorolactate in reactions with absolute requirements for divalent cation activators. 31P NMR analysis of the reaction products indicates that phosphorylation occurs at the alpha-position to generate the corresponding O- or S-bridged phosphate monoesters. In addition, the enzymes catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyzes the bicarbonate-dependent phosphorylation of fluoride ion. The kappa cat values for these substrates are 20-1000-fold slower than the kappa cat for oxaloacetate. Pyruvate and beta-hydroxypyruvate are not phosphorylated, since the enzyme does not catalyze the enolization of these compounds. Oxalate, a structural analogue of the enolate of pyruvate, is a competitive inhibitor of phosphoenolpyruvate carboxykinase (Ki of 5 microM) in the direction of phosphoenolpyruvate formation. Oxalate is also an inhibitor of the chicken liver enzyme in the direction of oxaloacetate formation and in the decarboxylation of oxaloacetate. The chicken liver enzyme is inhibited by beta-sulfopyruvate, an isoelectronic analogue of oxaloacetate. The extensive homologies between the reactions catalyzed by phosphoenolpyruvate carboxykinase and pyruvate kinase suggest that the divalent cation activators in these reactions may have similar functions. The substrate specificity indicates that phosphoenolpyruvate carboxykinase decarboxylates oxaloacetate to form the enolate of pyruvate which is then phosphorylated by MgGTP on the enzyme.

AMP nucleosidase: kinetic mechanism and thermodynamics.

The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular location of phosphoenolpyruvate carboxykinase in hepatocytes from fed and starved rats.

To evaluate published indications that about 25% of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is located in mitochondria of adult rat liver, cell fractionations were conducted with hepatocytes isolated from rats that were fed ad libitum or starved for 2 days. Hepatocytes were exposed to digitonin for 10 s, and the released materials were separated from residual cell structures by centrifugation through a layer of brominated hydrocarbon. In addition to PEPCK, activities of 9 other enzymes were measured in the untreated cells and with good recovery in the two fractions obtained with digitonin treatment. By comparison with the release of marker enzymes for the cytosol and mitochondria, the subcellular distribution of PEPCK was determined. With cells from either fed or 2-day-starved rats, this enzyme was released exactly like lactate dehydrogenase and within 2-3% of phosphoglycerate kinase and pyruvate kinase. These results indicate that, even after induction by starvation, at least 97% of PEPCK activity is located in the cytosol of rat liver.

Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue.

Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.