Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group

The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn+) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the Km value for agmatine (from 1.1 +/- 0.2 mM to 6.3 +/- 0.3 mM) and the Ki value for competitive inhibition by Gdn+ (from 15.0 +/- 0.1 mM to 44.2 +/- 2.1 mM), but the Ki value for putrescine inhibition (2.8 +/- 0.2 mM) was not altered. The tryptophan fluorescence properties (lambdamax = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 --> Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.     

Identification of human glutaminyl cyclase as a metalloenzyme. Potent inhibition by imidazole derivatives and heterocyclic chelators

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 +/- 1 and 295 +/- 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 +/- 0.02, 6.93 +/- 0.03, and 5.60 +/- 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

Study of the inhibition of neutral phosphatase from Pseudomonadaceae by derivatives of its substrates

The inhibition of neutral phosphatase isolated from the bacteria of the Pseudomonadaceae family by various fragments of the enzyme-hydrolyzed R-O-PO3H2 substrates, inorganic orthophosphate (KH2PO4) and its analogs as well as by adenine, adenosine, alcohols, sugars and amino acids, was studied. It was demonstrated that among other compounds tested only the orthophosphoric acid anions (H2PO4-) exhibit the properties of strong associative inhibitors (K1Vi = 4.35.10(-6)M of the enzyme. The pH dependence of the Michaelis constant [pKm0 = f(pH)] and the inhibition constant for phosphatase by potassium orthophosphate [pK1Vi(KH2PO4) = f(pH)] was studied. The presence in the enzyme active center of a carboxylic (pK = 4.3 +/- 0.1) (presumably, glutamine) and an imidazole (pK = 7.15 +/- 0.1) amino acid residues was postulated. The data obtained were compared to those for neutral, alkaline and acid phosphatases.     

The mechanism of action of dipeptidyl aminopeptidase. Inhibition by amino acid derivatives and amines; activation by aromatic compounds.

A variety of amino acid and peptide amides have been shown to be inhibitors of dipeptidyl aminopeptidase. Among these compounds derivatives of strongly hydrophobic amino acids are the strongest inhibitors (Phe-NH2, Ki = 1.0 +/- 0.2 mM), while amides of basic amino acids were somewhat less effective (Lys-NH2, Ki = 36 +/- 3 mM). Short chain amino acid amides are notably weaker inhibitors (Gly-NH2, Ki = 293 +/- 50 mM). The interaction of the side chains of compounds with the enzyme appears to be at a site other than that at which the side chain of the amino-penultimate residue of the substrate interacts since the specificity of binding is different. Primary amines have been shown to inhibit, e.g., butylamine, Ki = 340 +/- 40 mM, and aromatic compounds have been shown to stimulate activity toward Gly-Gly-NH2 and Gly-Gly-OEt (phenol, 35% stimulation of activity at a 1:1 molar ratio with the substrate). The data suggest that inhibition involves binding at the site occupied by the free alpha-amino group and the N-terminal amino acid.     

Reversible inhibitors of beta-glucosidase

A variety of reversible inhibitors of sweet almond beta-glucosidase were examined. These included simple sugars and sugar derivatives, amines and phenols. With respect to the sugar inhibitors and, indeed, the various glycoside substrates, the enzyme has what can be considered a "relaxed specificity". No single substituent on glucose, for example, is essential for binding. Replacement of a hydroxyl group with an anionic substituent reduces the affinity while substitution with a cationic (amine) substituent enhances the affinity. Amines, in general, are good inhibitors, binding more tightly than the corresponding alcohols: pKiRNH3+ = 0.645pKiROH + 1.77 (n = 9, r = 0.97). The affinity of a series of 10 primary amines was found to be strongly influenced by substituent hydrophobicity: pKi = 0.52 pi + 1.32 (r = 0.95). The major binding determinant of the glycoside substrates is the aglycon moiety. Thus, the Ki values of phenols are similar in magnitude to the Ks values of the corresponding aryl beta-glucoside. The pH dependence for the inhibition by various phenols indicates that it is the un-ionized phenol which binds to the enzyme when an enzymic group of pKa = 6.8 (+/- 0.1) is protonated. The affinity of the phenol inhibitor is dependent on its basicity with a Br?nsted coefficient for binding of beta = -0.26 (n = 14, r = 0.98). The pH dependence of the binding of two particularly potent beta-glucosidase inhibitors was also examined. 1-Deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol) has a pH-corrected Ki = 6.5 microM, and D-glucono-1,5-lactam has a pH-corrected Ki = 29 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.     

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.     

消炎痛对猪胃H~＋／K~＋－ATP_（ase）抑制的动力学

消炎痛作为一种可引起胃粘膜急性病变的药物,用分离提纯的猪胃Ｈ＋／Ｋ＋－ＡＴＰａｓｅ证明,它可以显著的抑制此酶的活力,０．１ｍｇ／ｍＬ时即可抑制酶活力２７％,０．５ｍｇ／ｍＬ时可抑制全部活力,其Ｋ（０．５）为０．１８ｍｇ／ｍＬ。消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制随３０℃时预保温时间的延长而加剧,１０ｍｉｎ预保温可抑制总活力的５０％。消炎痛并不影响Ｈ＋／Ｋ＋－ＴＡＰａｓｅ的转换温度（３９℃）以及最适ｐＨ（约ｐＨ７．５）,但酸性条件下消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ抑制比碱性条件下强烈。在我们的实验条件下,消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制与Ｈ＋／Ｋ＋－ＡＴＰａｓｅ量成正比,它不影响酶的Ｋｍ值（０．１１ｍｍｏｌ／Ｌ）,而是显著降低Ｖｍａｘ,因而它是此酶的可逆性非竞争性抑制剂,其Ｋｉ为０．３２ｍｍｏｌ／Ｌ。     

Aspartyladenylate analog--effective inhibitor of asparagine synthetase

A number of earlier unknown phosphonate analogues of aspartyl adenylate with anhydride oxygen substituted by --CH2--, and the carbonyl group substituted by --CH(OH)- or --CH(NH2)-groups were synthesized. These compounds were used to study the reaction mechanism of asparagine synthetases from white lupine and E. coli. The aspartyl adenylate analogues proved to be powerful competitive inhibitors (Ki = 10(-7) M) of the bacterial enzyme. In the case of white lupine enzyme catalyzing the aspartate-independent ATP--[32P]PPi exchange, the above compounds displayed a non-competitive type of inhibition with respect to aspartate and ATP, Ki = 10(-4) M. It is likely that for the latter enzyme the first intermediate is different from an aspartyl adenylate derivative.     

Active-site-directed inactivators of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.     

Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling.

Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was reduced to about 5% of wild-type activity. Tryptophan emission fluorescence (lambdamax = 340 nm), and CD spectra were nearly identical for wild-type and D153N agmatinases. The Km value for agmatine (1.6 +/- 0.1 mm), as well as the Ki for putrescine inhibition (12 +/- 2 mm) and the interaction of the enzyme with the required metal ion, were also not altered by mutation. Three-dimensional models, generated by homology modelling techniques, indicated that the side chains of Asp153 and Asn153 can perfectly fit in essentially the same position in the active site of E. coli agmatinase. Asp153 is suggested to be involved, by hydrogen bond formation, in the stabilization and orientation of a metal-bound hydroxide for optimal attack on the guanidinium carbon of agmatine. Thus, the disruption of this hydrogen bond is the likely cause of the greately decreased catalytic efficiency of the D153N variant.     

Inhibition of the intestinal transport of uracil by hexoses and amino acids

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.     

The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

Diguanosinetetraphosphatase from rat liver: Acitivity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate.

The hydrolysis of diadenosine tetraphosphate, a compound previously described by others to occur in liver at concentrations of around 0.1 mu M, is carried out by a specific enzyme. This enzyme has been partially purified from rat liver extracts, and the following properties have been found. The Km value for diadenosine tetraphosphate is 2 mu M; the products of hydrolysis are ATP and AMP; the Km value for diguanosine tetraphosphate is 2 mu M; none of the following substances were substrates of the enzyme: diadenosine triphosphate, diguanosine di and triphosphates, adenosine tetraphosphate, ATP, ADP, NAD+, NADP+ and bis-p-nitrophenylphosphate. Cyclic AMP was not an inhibitor of the reaction. The enzyme requires Mg2+ ions, is maximally active at a pH value of approximately 8, and has a molecular weight of 22000 as estimated by filtration on Sephadex G-100. The activation energy of the reaction was of 10250 cal times mol-1 (42886 J times mol-1). Particularly striking is the inhibition by adenosine tetraphosphate (Ki equals 48 nM) and guanosine tetraphosphate (Ki equals 14 nM). Other nucleotides tested were also competitive inhibitors with Ki values in the 10--100 mu M range.     

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., Lindstr?m, 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)     

Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.     

Inhibition of rat liver steroid 5 alpha-reductase by 3-androstene-3-carboxylic acids: mechanism of enzyme-inhibitor interaction

The interactions of a series of newly discovered inhibitors of delta 4-3-oxo-steroid 5 alpha-reductase (SR; EC 1.3.1.30), the 3-androstene-3-carboxylic acids (steroidal acrylates), have been studied by using a solubilized rat liver enzyme preparation. As exemplified by one member of this series, 17 beta-[N,N-diisopropyl-carbamoyl)androst-3,5-diene-3-carboxylic acid (1a), the dead-end inhibition patterns of selected compounds in this class are best evaluated by a linear uncompetitive kinetic model versus either substrate, testosterone (T) or NADPH. These results were interpreted within the context of the preferentially ordered kinetic mechanism for rat liver SR to arise from the association of inhibitor to the binary complex of enzyme and NADP+. This proposed inhibition mechanism was supported by data from double-inhibition experiments implicating the synergistic binding of steroidal acrylate and NADP+ to SR. Further evidence for the preferential formation of this ternary complex was obtained from filtration binding assays with [3H]-1a, where radioligand association to protein was greatly enhanced in the presence of NADP+. The amount of [3H]-1a binding to protein was proportional to the specific activity of SR in the enzyme preparations, and the estimated dissociation constant from binding data by Scatchard analysis (Kd = 25 nM) was comparable to the inhibition constants estimated for SR activity (Ki = 12-26 nM). From the pH profile for inhibition of the solubilized liver SR with 1a, it is proposed that the anion of the steroidal acrylate (pK1 = 4.7 +/- 0.2) is the active inhibitory species, coordinating to a protonated active site functionality (pK2 = 7.5 +/- 0.1). On the basis of data from similar experiments with structural analogues of 1a, the determinants for binding recognition and inhibitory potency are compared to structural features of the putative enzyme-bound intermediate states. These compounds represent a potential therapeutic alternative in the treatment of 5 alpha-dihydrotestosterone specific androgen dependent disease states.