Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.     

Comparison of angiotensinogen and tetradecapeptide as substrates for human renin. Substrate dependence of the mode of inhibition of renin by a statine-containing hexapeptide

The kinetic properties of two different substrates for human renin, a synthetic tetradecapeptide and the natural substrate human angiotensinogen, have been compared. While the Vmax was similar for the two substrates, the Km values differed by a factor of 10, i.e., 11.7 +/- 0.7 microM (tetradecapeptide) and 1.0 +/- 0.1 microM (angiotensinogen). The mode of inhibition of renin by a statine (Sta)-containing hexapeptide, BW897C, that is a close structural analog of residues 8-13 of human angiotensinogen (Phe-His-Sta-Val-Ile-His-OMe), was determined for the two substrates. Competitive inhibition was observed when tetradecapeptide was the substrate (Ki = 2.0 +/- 0.2 microM), but a more complex mixed inhibition mode (Ki = 1.7 +/- 0.1 microM, Ki' = 3.0 +/- 0.23 microM) was found with angiotensinogen as substrate. This mixed inhibition probably results from the formation of an enzyme-inhibitor-substrate or enzyme-inhibitor-product complex and reflects the more extensive interactions that the protein angiotensinogen, as opposed to the small tetradecapeptide substrate, can make with renin. We conclude that the mixed inhibition observed when angiotensinogen is used as renin substrate could be important in the clinical application of renin inhibitors because it is less readily reversed by increased concentrations of substrate than is simple competitive inhibition.     

Mechanistic and kinetic studies of inhibition of enzymes

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as nu/(V0 - nu) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V0 and nu are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing-substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, Km, Ki, Ki', and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.     

The pH dependence of binding of inhibitors to bovine adrenal tyrosine hydroxylase

The inhibition of purified bovine adrenal tyrosine hydroxylase by several product and substrate analogues has been studied to probe the kinetic mechanism. Norepinephrine, dopamine, and methylcatechol are competitive inhibitors versus tetrahydropterins and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine is an uncompetitive inhibitor versus tetrahydropterins and a competitive inhibitor versus tyrosine. The Ki value for 3-iodotyrosine depends on the tetrahydropterin used. These results are consistent with tetrahydropterin binding first to the free enzyme followed by binding of tyrosine. 5-Deaza-6-methyltetrahydropterin is a noncompetitive inhibitor versus tetrahydropterins and tyrosine. The effect of varying the concentration of tyrosine on the Ki value for 5-deaza-6-methyltetrahydropterin is consistent with the binding of this inhibitor to both the free enzyme and to an enzyme-dihydroxyphenylalanine complex. Dihydroxyphenylalanine also is a noncompetitive inhibitor versus tetrahydropterins and tyrosine; the effect of changing the fixed substrate is consistent with the binding of this inhibitor to both the free enzyme and to the enzyme-tetrahydropterin complex. The effect of pH on the Ki values was determined in order to measure the pKa values of amino acid residues involved in substrate binding. Tight binding of catechols requires that a group with a pKa value of 7.6 be deprotonated. Binding of 3-iodotyrosine involves two groups with pKa values of 7.5 and about 5.5, one of which must be protonated for binding. Binding of 5-deaza-6-methyltetrahydropterin requires that a group on the free enzyme with a pKa value of 6.1 be protonated. The Ki value for dihydroxyphenylalanine is relatively insensitive to pH, but the inhibition pattern changes from noncompetitive to competitive above pH 7.5, consistent with the measured pKa values for binding to the free enzyme and to the enzyme-tetrahydropterin complex.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

Cholecystokinin activation: evidence for an ordered reaction mechanism for the tyrosyl protein sulfotransferase responsible for the peptide sulfation.

The kinetics of the forward tyrosyl protein sulfotransferase (TPS) reaction were examined using an assay based on the 35SO4 transfer from 3'-phosphoadenosine 5'-phospho(35S)sulfate [( 35S]PAPS) to tyrosyl residues of the non-sulfated cholecystokinin derivative, BocCCK-8(ns). TPS present in the microsomal membranes from rat cerebral cortex was used for these studies. Initial velocity measurements performed over a wide range of PAPS, BocCCK-8(ns), 3'-PAP and BocCCK-8(s) concentrations, indicated that the reaction follows an ordered mechanistic pathway. The KM value determined for BocCCK-8(ns) was 160 +/- 18 microM, and that for [35S]PAPS was 0.15 +/- 0.03 microM. 3'-Phosphoadenosine 5'-phosphate (3'-PAP) was found to be a product inhibitor with a Ki = 0.30 +/- 0.02 microM. BocCCK-8(s) produced an uncompetitive inhibition pattern on the TPS reaction. Adenosine 5'-phosphosulfate (APS) behaved as a competitive inhibitor versus PAPS with a Ki = 3.0 +/- 0.3 microM. ATP inhibited competitively the reaction when PAPS was the varied substrate with a Ki = 3.6 +/- 0.5 microM. The results of product and substrate inhibition studies and the patterns of dead end inhibition obtained with APS are best fit by an ordered Bi-Bi reaction mechanism where PAPS is the first substrate to bind and 3'-PAP is the last product to be released.     

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3β-hydroxysteroid oxidoreductase (3β-HSD) from human placental homogenates were studied invitro. The apparent Michaelis constants for 3β-HSD with the substrates pregnenolone (Δ⁵P) and dehydroepiandrosterone (DHA) were 170 nM and 40 nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for pregnenolone was 20 μM and for DHA, 17 μM. The activity of 3β-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17β (Ki=46 nM), cholesterol (Ki=0.68 μM) and 16α-hydroxydehydroepiandrosterone (16αOHDHA) (Ki=2.2 μM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17β (Ki=69 nM), pregnenolone (Ki=91 μM), cholesterol (Ki=1.3 μM) and 16αOHDHA (Ki=1.9 μM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (Δ⁴P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.     

Substrate inhibition of Pseudomonas aeruginosa elastase by 3-(2-furyl)acryloyl-glycyl-L-phenylalanyl-L-phenylalanine

The kinetics of hydrolysis by Pseudomonas aeruginosa elastase at 37 degrees C and pH 7.3 of 3-(2-furyl)acryloyl-glycyl-L-phenylalanyl-L-phenylalanine is compatible with nonproductive substrate inhibition, i.e., v = V.[S]/(Km + [S] + [S]2/K1), and the values of Km, Ki, and kappa cat are 1.4 mM, 5.0 mM, and 240 s-1, respectively. Product inhibition experiments are in agreement with an ordered release of product, with L-phenylalanyl-L-phenylalanine, the amino-containing product, being released first from the elastase.product complex. The values of Ki for L-phenylalanyl-L-phenylalanine and 3-(2-furyl)acryloyl-glycine are 1.5 and 4.0 mM, respectively. Kinetic experiments indicate that the second molecule of substrate combines with elastase.substrate to form a dead-end elastase . (substrate)2 complex.     

Inhibition of cytochrome P450 mediated enzyme activity by alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effect on the enzyme. The obtained IC?? values of Cd2+, Ni2+, and Zn2+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd2+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn2+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.     

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.     

Carbonyl sulfide inhibition of CO dehydrogenase from Rhodospirillum rubrum

Carbonyl sulfide (COS) has been investigated as a rapid-equilibrium inhibitor of CO oxidation by the CO dehydrogenase purified from Rhodospirillum rubrum. The kinetic evidence suggests that the inhibition by COS is largely competitive versus CO (Ki = 2.3 microM) and uncompetitive versus methylviologen as electron acceptor (Ki = 15.8 microM). The data are compatible with a ping-pong mechanism for CO oxidation and COS inhibition. Unlike the substrate CO, COS does not reduce the iron-sulfur centers of dye-oxidized CO dehydrogenase and thus is not an alternative substrate for the enzyme. However, like CO, COS is capable of protecting CO dehydrogenase from slow-binding inhibition by cyanide. A true binding constant (KD) of 2.2 microM for COS has been derived on the basis of the saturable nature of COS protection against cyanide inhibition. The ability of CO, CO2, COS, and related CO/CO2 analogues to reverse cyanide inhibition of CO dehydrogenase is also demonstrated. The kinetic results are interpreted in terms of two binding sites for CO on CO dehydrogenase from R. rubrum.     

Kinetic properties of sheep brain glutathione reductase.

The kinetic properties of sheep brain glutathione reductase (GSSGR) were investigated. The enzyme showed Ping-Pong kinetics with double substrate inhibition in the forward direction. Km values for NADPH and GSSG were found to be 60.9 and 116.9 mumol/l, and Ki values were found to be 42.1 and 347.3 mumol/l, respectively. NADP+ inhibition at low fixed concentration of NADPH was mixed-type with a Ki of 281.5 mumol/l and alpha of 0.048. It is concluded that the enzyme shows a hybrid Ping-Pong-ordered branched mechanism.