N-Benzyloxycarbonyl-valyl-prolinal, a potent inhibitor of post-proline cleaving enzyme

A peptide aldehyde inhibitor possessing prolinal at the carboxyl terminus was designed as an inhibitor of post-proline cleaving enzyme by analogy with peptide aldehyde inhibitors of serine and thiol proteases. N-Benzyloxycarbonyl-valyl-prolinal was found to be a potent inhibitor of post-proline cleaving enzyme from ascidian sperm with a K1 value of 2.4 nM. The presence of the aldehyde portion of the inhibitor, as well as its prolonged incubation with the enzyme, is indispensable for the potent inhibitory activity of the inhibitor. These results indicate that N-benzyloxycarbonyl-valyl-prolinal functions as a transition-state aldehyde inhibitor of post-proline cleaving enzyme.     

Effect of alpha 1-proteinase inhibitor and sulphated polysaccharides on the activity of m beta-acrosin

Purified m beta-acrosin catalysed amidolysis in vitro of several p-nitroanilides with C-terminal arginine residues. alpha 1-proteinase inhibitor inhibited amidolysis catalysed by the enzyme. This effect of alpha 1-Proteinase inhibitor was not prevented by pre-incubation of the enzyme with heparin or any other glycosaminoglycan. Pre-incubation of the enzyme with sulphated dextran or sulphated cellulose alleviated the effect of alpha 1-proteinase inhibitor. These results are discussed in terms of possible in vivo modulation by alpha 1-proteinase inhibitor of acrosin activity.     

Inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.     

Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor

The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.     

Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.     

The proteinaceous inhibitor of limit dextrinase in barley and malt

Barley limit dextrinase catalyses hydrolysis of alpha-1,6-D-glucosidic bonds in branched poly- or oligosaccharides from starch. A specific inhibitor of this enzyme is found in mature barley kernels, but disappears after several days of germination. Two forms of this proteinaceous inhibitor, identical in amino acid sequence, have been isolated and characterized. They differ in attachment of cysteine or glutathione to a sulfhydryl group, possibly that of cysteine residue 59 of the inhibitor. They can form a 1:1 complex with limit dextrinase and are believed to interact specifically with the enzyme active site. The inhibitor present in mature barley can effectively reduce enzyme activity in barley germinated for a short time and in commercial malt.     

A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein

A thermostable NAD(P)⁺ glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.     

Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance

Saquinavir is a widely used HIV-1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV-1 viral enzymes. Three HIV-1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5-, 3-, and 419-fold increase in Ki values, respectively, compared to the wild-type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364-12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV-1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 A resolution. This mutant complex is compared with that of the wild-type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340-3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main-chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1' substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild-type enzyme and therefore may facilitate catalysis by the resistant mutant.     

The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.     

Purification and properties of the nuclease inhibitor of Aspergillus oryzae and kinetics of its interaction with crystalline nuclease O.

A nuclease inhibitor found in the mycelia of Aspergillus oryzae has been purified 158,000-fold by ammonium sulfate precipitation, chromatography on Sephadex G-75, DEAE-Sephadex A-50 and Bio-Gel p-60 columns, preparative disc electrophoresis on acrylamide gel, and electrofocusing in ampholite. The purified inhibitor is nearly homogeneous as judged by disc electrophoresis. It shows a typical ultraviolet absorption curve for protein, and the inhibitory activity is inactivated by chymotrypsin. The inhibitor and nuclease O (EC 3.1.4.9, a crystalline enzyme from the mycelia of the same organism) form a stable enzyme inhibitor complex. The molecular weights of nuclease O, the inhibitor and the enzyme inhibitor complex are estimated to be 46,000, 22,000 and 73,000 respectively, by Sephadex G-100 gel filtration. The isoelectric points of the enzyme and the inhibitor are 10.0 and 4.09, respectively, as determined by electrofocusing in ampholite. The inhibition is noncompetitive, and the inhibitor constant (K1) is 3.2 X 10(-12) M, whereas the Michaelis constant (Km) for DNA is 2.2 X 10(-8) M. The inactive enzyme-inhibitor complex is reactivated by chymotrypsin through inactivation of the inhibitor. The reactivated enzyme can be inactivated again by the inhibitor, which shows that desensitization of the enzyme does not occur by the action of chymotrypsin.     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Reaction of fungal amine oxidase with beta-bromoethylamine.

beta-Br-ethylamine is both a substrate and an irreversible inhibitor of amine oxidase from Aspergillus niger. The enzyme catalyzes the nonoxidative elimination of HBr from beta-Br-ethylamine to form acetaldehyde. beta-Br-ethylamine meets several criteria for an irreversible substrate analog or suicide inhibitor. 1) It inactivates the oxidized enzyme, but not the reduced enzyme. 2) The Michaelis constant for beta-Br-ethylamine in the elimination reaction showed a similar magnitude to that of the related constant found when the haloamine acted as an inhibitor. 3) The enzyme was protected from the inactivation by the co-existence of the substrate. 4) Inactivation with beta-Br-[14C]ethylamine resulted in the incorporation of radioactivity corresponding to 1 mol of the label/mol of the monomeric unit of the enzyme and a decrease of 1 mol of the -SH group. 5) Inactivation was accompanied by the formation of a new absorption peak at 320 nm which was bleached by addition of NaBH4.     

Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase

The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1. The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase. The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated. The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+. The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+. However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors. A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria. This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase. It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.     

Interaction of pig kidney and lentil seedling copper-containing amine oxidases with guanidinium compounds

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6

Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.     

Conversion of angiotensin-1 to angiotensin-2 by a latent endothelial cell peptidyl dipeptidase that is not angiotensin-converting enzyme

Cultured bovine pulmonary artery endothelial cells contain a second peptidyl dipeptidase, distinct from angiotensin-converting enzyme, present in an inactive form associated with a non-dialyzable inhibitor. Partial purification by glycine affinity chromatography separates enzyme from inhibitor to yield a preparation which hydrolyzes angiotensin-1, bradykinin, substance P, atriopeptin-2, enkephalin and Hip-His-Leu. This enzyme is resistant to inhibition by lisinopril, captopril, thiorphan, phosphoramidon, soybean trypsin inhibitor, PMSF and aminopeptidase and carboxypeptidase inhibitors, but is inhibited by EDTA.     

A peptide inhibitor of HIV-1 protease using alpha, beta- dehydro residues: a structure based computer model

HIV-1 encodes an aspartic protease, an enzyme crucial to viral maturation and infectivity. It is responsible for the cleavage of various protein precursors into viral proteins. Inhibition of this enzyme prevents the formation of mature, infective viral particles and therefore, it is a potential target for therapeutic intervention following infection. Several drugs that inhibit the action of this enzyme have been discovered. These include peptidomimetic inhibitors such as ABT-538 and saquinavir, and structure based inhibitors such as indinavir and nelfinavir. Several of these have been tested in human clinical trials and have demonstrated significant reduction in viral load. However, most of them have been found to be of limited clinical utility because of their poor pharmacological properties and also because the viral protease becomes rapidly resistant to these drugs on account of mutations in the enzyme. One way to overcome these limitations is to design an inhibitor that interacts mainly with the conserved residues of HIV-1 protease. By a rational drug design approach based on the high resolution X-ray crystal structure of the HIV-1 protease with--MVT 101 (a substrate based inhibitor) and the specific design principles of peptides containing dehydro-Alanine (delta Ala) derived from our earlier studies, we have designed a tetrapeptide with the sequence: NH2-Thr-delta Ala-delta Ala-Gln-COOH. Energy minimization and molecular modelling of the interaction of the designed tetrapeptide with the inhibitor binding site indicate that the inhibitor is in an extended conformation and makes excessive contacts with the viral enzyme at the interface between the protein subunits. The designed inhibitor has 33% of its interaction with the conserved region of HIV-1 protease which is of the same order as that of MVT 101 with the enzyme.     

Interaction of Pig Kidney and Lentil Seedling Copper-Containing Amine Oxidases with Guanidinium Compounds

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (K(i) =1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a K(i) value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.