A latent collagenase from embryonic human skin explants.

Collagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme. 1) Embryonic human skin explants showed a much higher capacity for collagenase production than did adult skin, on the basis of unit weight of tissue. 2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system. 3) The molecular weights of active and latent collagenases were about 40,000 and 50,000, respectively. 4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3 M. The degree of activation produced by this treatment was as high as that by limited proteolysis with trypsin. It was concluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex. 5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Binding of intrinsic ATPase inhibitor to mitochondrial ATPase--stoichiometry of binding of nucleotides, inhibitor, and enzyme

F1-ATPase inhibitor was purified from yeast, Saccharomyces cerevisiae. The purified inhibitor blocked ATPase activity in the presence of ATP and Mg2+ by forming a latent equimolar enzyme-inhibitor complex with ATP and ADP newly bound to loose sites on the enzyme. A small portion of externally added ATP was hydrolyzed before the latent complex was formed but the hydrolysis was not directly related to the complex formation. Newly bound ATP tended to be converted to ADP when the ATP concentration of the medium was low. ATP tightly bound to the enzyme was not directly involved in formation of the complex. The complex was fairly stable in the presence of excess inhibitor and ATP but at a high concentration of the enzyme (10(-5) M), the inhibition was not complete, although only about 0.03% of the original activity remained unblocked.     

Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2

EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.     

Higher-order complex formation between the 72-kilodalton type IV collagenase and tissue inhibitor of metalloproteinases-2.

The collagenases are a class of matrix degradative enzymes whose actions are important in physiological and pathological processes. The human 72-kDa type IV collagenase (matrix metalloproteinase-2) and its proteinase inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2), are produced as a proenzyme-inhibitor complex by numerous cell lines. We analyzed the quaternary structure of and enzyme-inhibitor interactions in the native enzyme-inhibitor complex by studying the pattern of complexes demonstrated by molecular weight determination in nondenaturing polyacrylamide gels and evaluating the products formed by reaction of the native complexes with cross-linking agents. Electrophoresis in native polyacrylamide gels demonstrates that approximately 79% of the latent enzyme is present in a 1:1 bimolecular complex with the inhibitor TIMP-2, with 21% present as a complete tetrameric complex of two molecules of collagenase combined with two molecules of TIMP-2. The enzyme complex activated with organomercurials displays a shift to a higher proportion of the bimolecular complex with only 5% present as higher molecular weight complexes. Cross-linking of the latent and active forms of the complex with bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) tartarate demonstrates both the 1:1 and 2:2 complexes as well as an intermediate form that appears to be a complex composed of two molecules of collagenase and one of TIMP-2. The distribution of cross-linked products is unchanged with the addition of excess TIMP-2 to the reaction mix, implying that the binding sites for TIMP-2 to the initial enzyme-inhibitor complex are all occupied when the stoichiometry is 1 to 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of irreversible enzyme inhibition: Co-operative effects

A mathematical treatment of multiple irreversible enzyme-inhibitor binding is presented. The model considered consists of an enzyme molecule with one active site, and with two inhibitor binding sites. Equations are derived which describe the dependence of the concentration of active enzyme species, as well as the concentration of irreversibly enzyme bound inhibitor, on the rate constants characteristic of the various enzyme-inhibitor complex species.     

On the environment of zinc in beef heart cytochrome c oxidase: an x-ray absorption study

The role of zinc in beef heart cytochrome c oxidase has been studied by using x-ray absorption spectroscopy, zinc depletion and secondary structure predictions of subunits of beef heart cytochrome c oxidase. The stoichiometry of zinc in cytochrome oxidase has been determined in 35 different preparations and found to be one-half of copper (Cu:Zu = 2:1). Zinc is tightly bound to this enzyme and cannot be removed by dialysis against EDTA. However, zinc could be partially (up to 50%) depleted by treating the enzyme with either dipicolinic acid or by trypsin digestion. This partial depletion of zinc does not change the O2 uptake rate. X-ray absorption spectroscopy shows that the atom is in a distorted tetrahedral environment with mostly sulfur ligands. Since subunit VIa removed by the digestion removes about one-half the zinc, a possible binding site involves the two S sites present in that subunit with an appropriate folding in a structural role.     

Latent and active forms of collagenase in rat uterine explant cultures: regulation of conversion by progestational steroids

A latent collagenase, activatable by trypsin, has been identified in the culture media of postpartum rat uterus explants. Progesterone at a concentration of 25 × 10^?6m reduced the level of active collagenase by approximately 50%, whereas, total enzyme levels (active + latent) remained essentially constant during the first 3 days of culture. In addition, medroxyprogesterone acetate at a concentration of 1 × 10^?6m reduced active enzyme by approximately 75% while only small decreases in total enzyme were observed. After the third day of culture, total enzyme levels were also significantly decreased. These data suggest that during the first 3 days in culture the progestins prevent the conversion of latent collagenase to its active form. A fraction capable of promoting the activation of explant collagenase was detected in the culture medium and was partially separated from the collagenase. Progesterone (25 × 10^?6m) or medroxyprogesterone acetate (1 × 10^?6m) caused a 50 or 71% decrease, respectively, in the levels of the activator.     

Evidence that latent collagenases are enzyme-inhibitor complexes.

Specific collagenase from the culture media of various rabbit tissues and cells exists in active and latent forms. Latent collagenase is most effectively activated with 4-aminophenylmercuric acetate, a thiol-blocking reagent, strongly suggesting that latent forms are enzyme-inhibitor complexes. A collagenase inhibitor from bone cultures, which may be closely related to the inhibitor of such latent enzyme complexes, was partially characterized.     

Evidence for a single active site in the human angiotensin I-converting enzyme from inhibitor binding studies with [3H] RU 44 403: role of chloride

Binding of the potent radiolabelled competitive inhibitor 3H RU 44403 to pure human kidney angiotensin-I converting enzyme was examined in equilibrium and non equilibrium conditions. Equilibrium dialysis experiments indicate that, despite the duplicated structure of the enzyme and the presence of two putative active sites, 3H RU 44403 interacts with a single high affinity (Kd = 0.44 +/- 0.05 x 10-9 M, n = 3) binding site. This suggests that only one of the two putative active sites is functional, and can bind substrates or inhibitors. Sodium chloride plays an essential role in the enzyme-inhibitor interaction. The formation of the complex is only slightly influenced by NaCl, but the kinetic of dissociation is dramatically dependent on NaCl concentration. In a Nacl free medium the complex is unstable and dissociates rapidly. These results are consistent with the hypothesis that chloride ion influences isomerization of the complex toward a more stable form.     

Inhibition of factor Xa by a peptidyl-alpha-ketothiazole involves two steps. Evidence for a stabilizing conformational change.

Recently, peptidylketothiazoles have been shown to be potent inhibitors of proteases, but the details of the interaction have not yet been studied. In the work presented here, the interaction of factor Xa, a coagulation protease, with the transition state inhibitor BnSO(2)-D-Arg-Gly-Arg-ketothiazole (C921-78) is characterized. C921-78 is a tight and selective inhibitor of the coagulation protease factor Xa (K(d) = 14 pM). The hydrolytic activity of factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-limiting step of the bimolecular combination of inhibitor and enzyme was competitive with the substrate. Conversely, the inhibitor could be displaced from the active site of the enzyme after exposure of the preformed complex to an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921-78-factor Xa complex resulted in a 60% increase in the magnitude of the fluorescence emission spectrum. Rapid mixing of the enzyme and inhibitor produces a monophasic fluorescence increase, compatible with spectral transition in a single step. The rate constant for this reaction increased hyperbolically with the concentration of C921-78, but the amplitude remained constant. These results are consistent with the initial formation of an enzyme-inhibitor complex (EI), followed by a unimolecular conversion of EI to EI linked to a spectral transition. The rate constants of the isomerization provide an estimate of 300000-fold stabilization. Thus, the inhibition of factor Xa by C921-78 follows a mechanism similar to that described classically for slow tight binding inhibitors. However, the two steps of the reaction cannot be kinetically separated by the rapid equilibrium assumption, and therefore, the formation of EI is partially rate-limiting, too. The driving energy for the unusually fast isomerization step may result from the highly favorable interactions of the inhibitor in the primary binding site.     

Partial purification and characterization of exudate gelatinase in the acute phase of carrageenin-induced inflammation in rats

Gelatinase has been partially purified from exudate in the acute phase of carrageenin-induced inflammation in rats. The enzyme occurs in a latent form that can be activated with 4-aminophenylmercuric acetate (APMA). The latent gelatinase was separated into an active gelatinase and a protein fraction by zinc-chelating Sepharose 6B column chromatography in the final step of purification, suggesting that the latent gelatinase is an enzyme-inhibitor complex. The pH optimum of the active gelatinase is about 7.5 and no reactivity toward native type I collagen or alpha-casein was detected. The molecular weights of the latent and active gelatinases were about 245,000 and about 185,000, respectively, as determined by gel filtration on Sephadex G-200. On the other hand, both latent and active gelatinases occurred in multiple forms in SDS-substrate polyacrylamide gel electrophoresis; the latent gelatinase showed two bands with molecular weights of 105,000 and 69,000, and two additional bands of 88,000 and 83,000 appeared when the latent gelatinase was activated with APMA, while the active gelatinase showed all four species. The active gelatinase was inhibited by metallo-proteinase inhibitors, but not by serine- or cysteine-proteinase inhibitors, suggesting that the exudate gelatinase is a metallo-proteinase. The active gelatinase was also inhibited by serum proteins such as albumin and gamma-globulin, suggesting that gelatinase does not remain in an active form in the inflammatory lesion, where the vascular permeability is increased.     

Characterization of activatable form of prolyl hydroxylase in L929 fibroblasts

Prolyl hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase, EC 1.14.11.2) activity in a sonicated preparation of early log-phase L929 cells could be increased 3-4-times by preincubation of the sonicate with all cofactors of proline hydroxylation, such as ascorbate, Fe2+ and alpha-ketoglutarate. An "activatable" form of the enzyme is produced in these cells due to a deficiency of one of the cofactors in these cultures. The activatable form is found to be different from the active enzyme with respect to its stability to heat and dithiothreitol denaturation. The activatable form has different ionic properties and could be separated from the active enzyme by DEAE-Sephadex chromatography. The available evidence suggests that the activatable form is a tight complex produced by the enzyme with underhydroxylated collagen, the latter being produced by a cofactor deficiency in the cells. Activation of this complex follows the hydroxylation of the substrate and its subsequent release from the bound enzyme.     

Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

Kinetic studies of the alkaline mesentericopeptidase. Study on the active centre topography of alkaline mesentericopeptidase by means of bifunctional reversible inhibition.

The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n=2-8) and Ph(CH2)n-B(OH)2, (n=0-4), on the alkaline mesentericopeptidase-catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKa of about 6.9-7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme-inhibitor complex. The dependence of the degree of the enzyme-inhibitor complex formation upon the length of the side-chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.     

Leukotriene A4 hydrolase: a zinc metalloenzyme

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

The latency of spinach chloroplast phenolase

The latent phenolase in spinach chloroplast membranes could be activated by treatment with various detergents. Examination by thin-layer gel filtration showed the presence of two active proteins (one with lower MW called protein A and the other, protein B). The protein B was converted to A by dilution or on standing, and the latter conversely to the former by concentration. On freezing, an extract of the acetone powder of the chloroplasts, phenolase activity was strikingly reduced, and this is ascribed to an association of the protein A and a low MW (diffusible) substance giving rise to an inactive enzyme-inhibitor complex. The activity declined from autumn to winter, and it appears that the second type of latency due to the formation of the above complex is also involved.     

Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase

Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I-collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma-anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH.