Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.     

Purification and characterization of a metalloprotease from Chryseobacterium indologenes Ix9a and determination of the amino acid specificity with electrospray mass spectrometry

The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.     

The binding activity of estrogen receptor to DNA and heat shock protein (Mr 90,000) is dependent on receptor-bound metal

1,10-Phenanthroline inhibited the DNA-cellulose binding of the transformed calf uterus estrogen receptor (homodimer of 66-kDa molecules: 5 S estrogen receptor) in a temperature- and concentration-dependent manner. This result appears related to the metal-chelating property of 1,10-phenanthroline, since the inhibition was decreased by addition of Zn2+ and Cd2+, but not by Ca2+, Ba2+, or Mg2+ for which the affinity of the chelator is low. Only a slight inhibition was observed in the presence of the 1,7-phenanthroline, a nonchelating analogue. After dialysis or filtration to remove free 1,10-phenanthroline, DNA binding of the 5 S estrogen receptor was still inhibited. Conversely, the chelator was unable to release prebound 5 S estrogen receptor from DNA-cellulose. The 5 S estrogen receptor DNA binding was inhibited when 1,10-phenanthroline was present during the transformation to activated receptor of the hetero-oligomeric nontransformed 9 S estrogen receptor, in which the hormone binding subunits are associated with heat shock protein, Mr 90,000 (hsp 90) molecules. In contrast, if 1,10-phenanthroline was removed before the transformation took place, only a slight inhibition was observed. Other experiments with EDTA indicated a similar inhibition of DNA-cellulose binding by the 5 S estradiol receptor, and all metal ions chelated by this agent prevented its inhibitory effect. The results indicate that 1,10-phenanthroline inhibited the DNA binding of the transformed 5 S estradiol receptor by chelating metal ion tightly bound to the receptor, which is not accessible to the chelator when the receptor is bound to DNA or to hsp 90. Therefore, they suggest that the metal ion may play a critical role in the interaction with DNA and hsp 90 by maintaining the structural integrity of the implicated receptor domain.     

Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding beta-ureido acids. Previous work has shown that incubation of this Zn2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226, 469-483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid greater than o-phenanthroline greater than 2,6-dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is well-described by ionization of a single group with a pK of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both Vmax and the apparent affinity for substrate. Conversely, binding of substrate decreases the pK of this group to about 5. L-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of L-dihydroorotic acid increases the pK of the ionizable group to 6.5. The agreement between the pK in the enzyme-L-dihydroorotic acid complex and the higher pK observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of acid, and chelators. The different effects of substrate and L-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.     

The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.     

Characterization of the zinc binding site of bacterial phosphotriesterase.

The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.     

Influence of EDTA and metal ions on a metalloproteinase from Pseudomonas fluorescens biotype I

The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.     

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+.     

L-histidinol dehydrogenase, a Zn2+-metalloenzyme

The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.     

Zinc irreversibly damages major enzymes of energy production and antioxidant defense prior to mitochondrial permeability transition

Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, alpha-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 microm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.     

Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo.

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4 degrees C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.     

Bovine liver dihydropyrimidine amidohydrolase: purification, properties, and characterization as a zinc metalloenzyme

Beef liver dihydropyrimidine amidohydrolase has been purified to homogeneity using both an electrophoretic and a hydrophobic chromatographic method. The enzyme is a tetramer with a molecular weight of 226,000 g mol-1, a subunit molecular weight of 56,500 g mol-1, and contains 4 mol of tightly bound (Ks greater than or equal to 1.33 X 10(9) M-1) Zn2+ per mole of active enzyme. The enzyme appears to be a true Zn2+ metalloenzyme because there exists a direct proportionality between enrichment of Zn2+ and active enzyme during purification, there is an almost quantitative relationship between the loss of both enzyme activity and Zn2+ during 8-hydroxyquinoline-5-sulfonic acid treatment to form apoenzyme, Zn2+ and Co2+ reactivate dipicolinic acid-inhibited enzyme, and saturating concentrations of a substrate, dihydrothymine, protect against 8-hydroxyquinoline-5-sulfonic acid inhibition. EDTA does not inhibit the enzyme; however, 8-hydroxyquinoline-5-sulfonic acid, o-phenanthroline, and 2,6-dipicolinic acid cause a time-dependent loss in activity which follows pseudo-first-order kinetics. Analysis of the resulting kinetic data for these three chelators indicates that the reaction pathway involves the formation of an enzyme-Zn2+-chelator ternary complex which then dissociates to form apoenzyme and a Zn2+-chelator complex. Like other Zn2+ metalloenzymes, the enzyme is inhibited by a number of substituted sulfonamides. In the case of p-nitrobenzenesulfonamide, this inhibition is competitive in nature. Using the purified enzyme, kinetic constants were determined for a variety of dihydropyrimidines, ureidocarboxylic acids, and hydantoin substrates. Normal hyperbolic kinetics were observed for the hydrolysis of the cyclic compounds, but the cyclization of the ureidoacids showed biphasic kinetics and different values of Km can be estimated at either high or low concentrations of these substrates.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Influence of 1,10-phenanthroline and its analogues, other chelators and transition metal ions on dipeptidase activity of the rumen bacterium, Prevotella ruminicola

Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu²⁺, Cr²⁺ and Hg²⁺ were most inhibitory, decreasing Ala₂ breakdown to 15, 15 and 5% of control activity, whereas Co²⁺, Mn²⁺ and Zn²⁺ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l⁻¹ and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo.     

Transthyretin is a metallopeptidase with an inducible active site

TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including β-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH?7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.     

State and accessibility of zinic in yeast alcohol dehydrogenase.

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited in the presence of 1,10-phenanthroline. 2. A conformational change in the enzyme's structure is induced by 1,10-phenanthroline, and is abolished in the presence of NADH. 1,10-Phenanthroline binds to the enzyme competitively with respect to NADH, with a stoicheiometry of 2 mol of 1,10-phenanthroline/144000g of enzyme. 3. 1,10-Phenanthroline induces a time-dependent dissociation of Zn2+ from the enzyme, which is in correlation with its inhibitions. 4. Spectrophotometric measurement indicates that the dissociation of half (2 zinc atoms/tetramer) of the total zinc content of the enzyme correlates with the full inhibition of its activity. Measurement of the tightly bound Zn2+ by atomic absorption photometry confirms this. 5. A proposition is advanced that the tetrameric molecule of yeast alcohol dehydrogenase possesses an inherent asymmetry, with four monomeric subunits being arranged in two mutually symmetrical pairs.     

Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures

Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.