Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rationale for the design of an inhibitor of tyrosyl kinase

Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.     

Potent inhibition of membrane-bound rat intestinal alkaline phosphatase by a new series of phosphate analogues.

The inhibition by phosphonates and phosphate analogues of the alkaline phosphatase activity of rat intestinal brush-border membrane vesicles was studied at pH 7.5 and 30 degrees C. Phenylene-1,3-diphosphonate, 2,6-dinitrophenylphosphonate and phosphonoacetaldehyde were found to be competitive inhibitors, with Ki values in the range 16-80 microM. Adenosine 5'-[beta-thio]diphosphate and adenosine 5'[gamma-thio]triphosphate are also very potent inhibitors, with Ki values of approx. 10 microM. The inhibition produced by these thiophosphates was mainly competitive but with a slight non-competitive element. Adenosine 5'-[beta gamma-imido]triphosphate is also a competitive inhibitor of the alkaline phosphatase, but oxidation of the ribose moiety of this compound with NaIO4 results in an active-site-directed irreversible inhibitor that could be of general use in studies of the mechanism of action of this enzyme.     

5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition of nucleotides.

5'-Nucleotidase prepared from muscle of small intesting of pig is strongly inhibited by nucleoside di- and triphosphates and their phosphonate analogs. Substrate kinetics appromate the Michaelis-Menten for for AMP, which shows a Km of 3-6 muM at pH 5.3-7.2. Inhibition is characterized as partial competitive, except at pH 5.3, where inhibition by ATP is noncompetitive. The Ki values for several inhibitors have been determined, and their departure from completeness of competitive inhibition has been studied. Inhibitor cooperativity of the type reported for the enzyme from sheep brain (P. L. Ipata (1968), Biochemistry 7, 507) was not observed for the enzyme from gut. In addition we failed to confirm sigmoid inhibition kinetics with 5'-nucleotidase from sheep brain.     

Purification, substrate specificity, and classification of tripeptidyl peptidase II

An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (B?l?w, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain

A synthetic peptide modeled after the calmodulin (CaM)-binding domain of rabbit skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys5-Lys-Asn-Phe-Ile-Ala10-Val-Ser-Ala-Ala-+ ++Asn15-Arg-Phe-Glycyl amide (M5), inhibited the CaM-independent chymotryptic fragment of the enzyme, C35 (Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), with a Ki of 3.2 +/- 2.1 microM. Inhibition was competitive with respect to the peptide substrate Lys-Lys-Arg-Ala-Ala5-Arg-Ala-Thr-Ser-Asn10-Val-Phe-Ala and was of the noncompetitive linear mixed type with respect to ATP. M5 and homologues with a serine residue substituted at positions 9, 13, or 14 were phosphorylated with the following order of preference: M5(Ser9) greater than M5(Ser13) much greater than M5(Ser14) greater than M5. The order of preference observed agreed with that predicted by comparison of the sequence of these peptides with the phosphorylation sites of myosin P-light chains. Both inhibition of C35 by M5 and phosphorylation of M5 and its serine-substituted homologues were severely curtailed by the addition of a stoichiometric excess of CaM over peptide. Thus, synthetic peptides modeled after the CaM-binding domain of skeletal muscle myosin light chain kinase can function as calmodulin-regulated active site-directed inhibitors of the enzyme.     

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [N?gler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.     

Histidine decarboxylase of Lactobacillus 30a. Hydroxylamine cleavage of the -seryl-seryl- bond at the activation site of prohistidine decarboxylase

Conversion of the pi subunit of prohistidine decarboxylase to the alpha beta subunits of the active enzyme proceeds by a nonhydrolytic, monovalent cation-dependent, serinolysis reaction in which the hydroxyl oxygen of serine 82 of the pi chain is incorporated into the carboxyl group at the COOH terminus (serine 81) of the beta chain. Serine-82 becomes the pyruvate residue at the NH2 terminus of the alpha chain (Recsei, P.A., Huynh, Q. K., and Snell, E.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977). The unusual reactivity of this particular -Ser-Ser- bond is demonstrated by its sensitivity to 1 M hydroxylamine, which cleaves the native proenzyme under mild conditions (pH 8.0, 37 degrees C) to yield a modified beta chain with serine hydroxamate at the COOH terminus (Ser-81) and a modified alpha chain containing serine (Ser-82 of the proenzyme) rather than pyruvate at the NH2 terminus. Neither an -Asn-Gly- bond nor other -Ser-Ser- bonds in the proenzyme were cleaved under these conditions. The reaction also did not occur with the denatured enzyme or with model peptides, indicating that the enhanced reactivity is a result of the particular conformation at this position in the native protein. The reaction with the native proenzyme proceeded optimally at pH 7.5-8.0 with a half-time (30 min) substantially less than that (3.5-4.5 h) required for the activation reaction and was not increased in rate by addition of K+. Correspondingly, preincubation of the proenzyme at pH 8.0 in the absence of both hydroxylamine and K+ modestly increased the rate of activation when K+ was subsequently added. Although these findings do not exclude other mechanisms, they are all consistent with and most easily explained by rearrangement of the pi chain to form an internal ester intermediate prior to the beta-elimination that occurs during activation to yield the alpha and beta chains of the mature enzyme.     

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.     

Metabolism of aromatic compounds by fungi. Kinetic properties and mechanism of 3-carboxy-cis,cis-muconate cyclase from Aspergillus niger.

A preliminary investigation of the kinetic properties of 3-carboxy-cis,cis-muconate cyclase (EC 5.5.1.5) has been performed. The initial velocity of the reaction was shown to be proportional to the concentration of the enzyme in the assay system adopted and the apparent Km was found to be 57 muM at pH 6.0 and 30 degrees C but at concentrations exceeding 70 muM, substrate inhibition was apparent. At pH 6.0 the Ki for the substrate was 0.45 mM. Plots of V and Km against pH showed inflexions at pH 5.3 and pH 6.4. The enzyme was inhibited by a variety of inorganic anions and by a number of dicarboxylic and tricarboxylic acids. The degree of inhibition exerted by these acids was found to be proportional to the proximity of their carboxyl groups, the cis configuration being a more effective inhibitor than the trans configuration. As inhibition was competitive in each case, the presence of an anion-sensitive substrate-binding site has been postulated. The cis-cis, cis-trans and trans-trans isomers of muconate, 3-chloromuconate and 3-carboxy-cis-trans-muconate, close analogues of natural substrate but not attacked by the enzyme, were also found to be competitive inhibitors. The variation in pKi with pH was determined in the case of cis,cis-muconate and cis-aconitate, both of which gave curves suggesting the importance of a group with a pKa of approximately 6.4 responsible for increasing the inhibition of the enzyme. Modification by ethoxyformic anhydride and the kinetics of Rose-Bengal-sensitized photo-oxidation suggested the participation of a histidine residue in the catalytic reaction. These results are discussed in the light of recent work on enzymes catalysing analogous reactions; a likely reaction mechanism has been proposed.     

Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups.

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.     

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide.

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate alpha-N-benzoyl-L-arginine ethl ester were studied in the region of pH 3.5-9.0. All substrates (and inhibitors) test, with the exception of alpha-N-benzyoyl-L-arginine p-nitroanilide, displayed approximately a two fold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is probably Trp-177.     

A recipe for designing water-soluble, beta-sheet-forming peptides.

Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates.     

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.     

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.     

alpha-Bromo ketone substrate analogues are powerful reversible inhibitors of carboxypeptidase A

The aldehyde (RS)-2-benzyl-4-oxobutanoic acid, which is 25% hydrated at pH 7.5, has recently been shown to be a strong reversible competitive inhibitor of carboxypeptidase A [Ki = 0.48 nM; Galardy, R. E., & Kortylewicz, Z. P. (1984) Biochemistry 23, 2083-2087]. The ketone analogue of this aldehyde (RS)-2-benzyl-4-oxopentanoic acid (IV) is not detectably hydrated under the same conditions and is 1500-fold less potent (Ki = 730 microM). The ketone homologue (RS)-2-benzyl-5-oxohexanoic acid (XIII) is also a weak inhibitor (Ki = 1.3 mM). The alpha-monobrominated derivatives of these two ketones are, however, strong competitive inhibitors with Ki's of 0.57 microM and 1.3 microM, respectively. Oximes derived from the aldehyde, the ketones IV and XIII, and a homologue of the aldehyde are weak inhibitors with Ki's ranging from 480 to 7900 microM. The inhibition of carboxypeptidase A by the alpha-monobrominated ketones is reversible and independent of the time (up to 6 h) of incubation of enzyme and inhibitor together. Bromoacetone at a concentration of 30 mM does not inhibit carboxypeptidase A. Incubation of an equimolar mixture of 2-benzyl-4-bromo-5-oxohexanoic acid (XV) and enzyme for 1 h led to the recovery of 82% of XV, demonstrating that it is the major species reversibly bound during assay of inhibition. Taken together, these results indicate that tight binding of carbonyl inhibitors to carboxypeptidase A requires specific binding of inhibitor functional groups such as benzyl and an electrophilic carbonyl carbon such as that of an alpha-bromo ketone or aliphatic aldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urease inhibition by polymer analogs of substrate and thiophosphamides

Inhibition of soybean urease by polymeric substrate analogues, urea and thiourea polydisulfides (PDSU and PDSTU, respectively), or three thiophosphoric acid amides (TPAA), tri-(N-3-hydroxyphenyl)thiophosphamide (1), tri-(N-4,4'-aminodiphenyl)thiophosphamide, and di-oxy-(N-alpha-piridyl)thiophosphamide (3) was studied in aqueous solutions at various pH values. The inhibitory effects of all these substances were reversible and competitive with the lowest inhibition constant Ki 2.8 microM for TPAA-1 at pH 3.85. Above and below this pH value, Ki increased reaching 24 [mu]M at pH 7.2. All test substances inhibited urease comparably with known inhibitors such as thiols (cysteamine, etc.) and hydroxamic acid derivatives, but were less efficient than phosphorodiamidates. Structural features of possible urease inhibitors of higher efficiency were proposed.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.