血管紧张素转换酶2-血管紧张素1-7-Mas轴对血管作用的研究进展

血管紧张素转换酶2（ACE2）和Mas受体的发现使人们对肾素-血管紧张素（RAS）有了更全面的认识。ACE2可水解血管紧张素Ⅰ和血管紧张素Ⅱ直接或间接生成血管紧张素1-7（Ang 1-7）,并与高血压的形成密切相关。Ang 1-7主要通过Mas受体引起血管舒张、抑制细胞增殖。ACE2-Ang1-7-Mas轴的发现为RAS的研究、高血压等心血管疾病的防治和新药开发提供了新的思路和方向。     

Zinc deficiency aggravates tubulointerstitial nephropathy caused by ureteral obstruction

Ureteral ligation causes tubulointerstitial nephropathy characterized by the tubular dilatation, the interstitial expansion, and a leukocyte infiltration into the tubulointerstitium. The present study was designed to explore whether zinc (Zn) deficiency affects the development of unilateral ureteral obstruction (UUO)-induced tubulointerstitial nephropathy. Compared to rats fed a standard diet, rats fed a Zn-deficient diet exhibited a greater influx of leukocytes into the tubulointerstitium of the cortex of the obstructed kidney 3 d following UUO. Furthermore, rats fed a Zn-deficient diet showed a slight invasion of leukocytes into the tubulointerstitium of the cortex of the contralateral nonobstructed kidney (CLK), although there was no infiltration of leukocytes into the CLK of rats fed a standard diet. These histological changes, however, were ameliorated by administration of enalapril, an angiotensin (ANG) I-converting enzyme inhibitor. Thus, it is suggested that Zn deficiency aggravates UUO-induced tubulointerstitial nephropathy via an increase in the action of ANG II.     

Relationship between plasma zinc, angiotensin-converting enzyme, alkaline phosphatase and onset of symptoms of zinc deficiency in the rat

Recent evidence suggests that changes in plasma zinc concentration may play a central role in the development of early lesions of zinc deficiency. The aim of the following work was to better understand events occurring in plasma during the onset of zinc deficiency, and to investigate biochemical mechanisms by which plasma zinc may exert its effects. Fifty male weanling rats of 90 g weight were allocated to five treatment groups of ten rats each. Treatments were: 1, zinc deficient, mixed diet (1-2 mg Zn per kg): 2, zinc deficient, self-select diet; 3, zinc repleted; 4, control, pair fed; 5, control, ad libitum fed. With the exception of treatment 1, which consisted of a 25% casein diet, all rats were offered protein as a separate component of the diet. Control rats received zinc in the drinking water (100 mg l-1). The sequence of events following initiation of zinc deficiency were: reduced plasma zinc concentration (2 days), reduced plasma angiotensin-converting enzyme and alkaline phosphatase activities (3-4 days), reduced feed intake and growth (5-6 days) and reduced percentage protein intake (12 days). Plasma zinc concentration in the deficient rats was inversely correlated with the growth rate of the rat over the previous 24 h. Zinc repletion resulted in marked overshoot in plasma zinc concentration (300%) and converting-enzyme activity (150%) within 24 h, but a return to normal within 72 h. Alkaline phosphatase activity responded likewise, albeit more slowly. Protein self selection had no effect on the manifestations of zinc deficiency, although reduced protein intake was associated with lower plasma zinc concentration. The results provide evidence of a role for plasma zinc in the development of early clinical signs of zinc deficiency, possibly acting biochemically through reduced activity of zinc-dependent peptidases such as angiotensin-converting enzyme.     

Metal ion effects on target sites of modification in metallocarboxypeptidase B

Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.     

Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.     

Binding of inhibitory metals to yeast enolase

Certain divalent cations can inhibit yeast enolase by binding at sites that are distinct from those metal binding sites normally associated with catalytic activity, i.e., the conformational and catalytic binding sites. By using a buffer that does not compete with metal ions (tetrapropylammonium borate) Zn, Co, Mn, Cu, Cd, and Ni are found to exhibit similar inhibitory characteristics. Inhibition by those metals is alleviated by the addition of imidazole or tris buffer and, for zinc, by a metal chelating agent (Calcein). Inhibition by zinc was examined in detail through binding studies and enzymatic activity measurement. In tetrapropylammonium buffers at pH 8.0, enolase binds up to four moles of zinc per mole of enzyme (two moles per subunit). An imidazole concentration of 0.05 M reduces the binding: in the absence of substrate, just two moles of zinc per enzyme are bound. The enzyme will bind two additional moles of zinc upon the addition of substrate in either buffer, but the enzyme in tetrapropylammonium buffer is nearly inactive. Inhibition is, therefore, correlated with the binding of two moles of zinc per mole of enzyme. Some additional metal ions, Ca, Tb, Hg, and Ag also caused inhibition of yeast enolase but not by binding to the inhibitory site described.     

Yeast RNA polymerase III: A zinc metalloenzyme

Atomic absorption spectroscopy has been used to demonstrate that zinc is associated with yeast RNA polymerase III. The enzyme purified by DNA-Sepharose chromatography gives a single predominant protein band in polyacrylamide gel electrophoresis and contains 0.7 gram-atoms of zinc per 100,000 grams of protein. The zinc is tightly associated with the enzyme and cannot be removed by passing the protein through a column of Chelex-100 resin under conditions where free zinc is quantitatively removed. Inhibition by the chelating agent 1,10-phenanthroline demonstrates that the zinc is essential to the catalytic process. The enzyme is inhibited 50% at 0.1 mM and 100% at 1 mM 1,10-phenanthroline.     

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc-sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC(50) value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.     

Properties of a cobalt-reactivated form of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (Y-ADH) is a widely studied metal-enzyme for its well-known biotechnological importance. Although its structure has been extensively investigated, some topics still remain controversial (zinc content and role), and various attempts aiming at modifying its structure to improve its catalytic properties have been made. In this paper, a metal-substituted Y-ADH has been prepared in vitro, in which one Zn atom per molecule (only one of those directly involved in catalysis) has been substituted by one Co atom. The substitution was obtained through zinc removal by a chelating treatment (with Chelex 100) followed by cobalt insertion. The zinc content in the native enzyme was preliminarily evaluated (taking care to avoid contamination) to be 4.1±0.1 g-at./molecule. After cobalt substitution, the ratio Zn:Co in the enzyme results to be 3:1. The active Co-Y-ADH has been compared with the native enzyme: it has lower specific activity (about 50%) and lower substrate affinity but greater thermo-resistance and a pH stability in a wider range than the native Y-ADH. A similar behavior, as far as cobalt content, thermo-resistance and pH stability are concerned, but greater specific activity and substrate affinity, were shown by an in vivo-substituted Co-Y-ADH obtained in a previous study.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Zinc and cadmium 5-aminolevulinate dehydratase. Metal-dependent pH profiles

Native 5-aminolevulinic acid dehydratase contains zinc ions, which are essential for the enzymatic activity. Replacement of zinc by cadmium yielded an active enzyme whose kinetic parameters (kkat and Km) are similar to those of the zinc enzyme in the neutral pH range. However, the pH profiles of kcat and Km were different due to different pKa values. Two groups both with pKa values of 6.5 in the free zinc enzyme, but with pKa values of 7.0 in the cadmium enzyme were calculated from plots of log (kcat/Km) versus pH. On the other hand, the enzyme-substrate complex is controlled by one acidic group (zinc pKa = 6.0, cadmium pKa = 6.4) and one basis group (zinc pKa = 8.2, cadmium pKa = 7.7) as calculated from plots of log kcat versus pH. The Arrhenius plots for kcat of the two enzymes show no significant difference, the free energies of activation are 77.1 kJ/mol for the zinc and 76.8 kJ/mol for the cadmium enzyme. From this and from previous work it is concluded that the metal ions are located near the active site and influence the ionisations of essential amino acid residues. From the pH profiles of the modifying reaction and inhibition by diethylpyrocarbonate a histidinyl residue is inferred as one of the ionisable groups of the active site.     

Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.     

Comparison of angiotensin-converting enzyme, malonaldehyde, zinc, and copper levels in preeclampsia

Preeclampsia is a syndrome of unknown etiopathogenesis. Recent studies carried out on preeclampsia have focused on the increase in free radicals in the feto-placental unit with poor perfusion. It is believed that the renin-angiotensin system (RAS) has a role in the poor perfusion of the placenta. It is uncertain whether there is a pre-existing impairment in RAS in pre-eclamptic pregnant women or not. In the present study, we measured angiotensin-converting enzyme (ACE), malonaldehyde (MDA), zinc, and copper levels in the placental tissue of 16 pre-eclamptic pregnant women and compared them with those in 20 healthy pregnant women. Whereas ACE activity and MDA were found to be high in the placentas of pre-eclamptic patients, zinc and copper levels were low and there was a negative correlation between ACE activity and zinc concentration. These findings suggest that high ACE activity might play a role in the increase in tissue hypoxia and consequent lipid peroxidation through vasoconstriction; zinc deficiency in the placental tissue might cause insufficiency of superoxide dismutase, an antioxidant enzyme. Furthermore, deficiency in placental zinc also plays a role in the biosynthesis of connective tissue, maintaining its integrity, which might have an impact on the structure of the spiral arteries     

The metal ion requirement for activation of latent collagenase from human polymorphonuclear leucocytes

Latent human PMN leucocyte collagenase (enzyme-inhibitor complex) was shown to require zinc for the property of being activatable by various disulfides [see Macartney, H.W. and Tschesche, H. (1980) FEBS Lett. 119, 327--332]. The active enzyme also requires zinc for activity, indicating a possible participation in the enzyme's reaction mechanism and/or stabilization of the active site. The zinc in the latent enzyme may be removed by dialysis against EDTA, or cysteine. This produces a zinc-free latent enzyme which cannot be activated by any of the disulfide-containing activators. Readdition of zinc to the EDTA-inhibited latent enzyme, at the same concentration as the EDTA, produces an activatable latent enzyme once again. However, excessive zinc concentrations (more than three times the concentration of EDTA) exhibited an inhibitory effect on the activation process. Thereafter the inhibitor cannot be removed by disulfides from the enzyme-inhibitor complex of the latent enzyme. The zinc in the latent enzyme may be replaced by other double-positive metal ions such as cobalt, manganese, magnesium and copper.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH on the modulation of rat osseous plate alkaline phosphatase by metal ions.

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.     

Two different zinc sites in bovine 5-aminolevulinate dehydratase distinguished by extended X-ray absorption fine structure

The zinc coordination in 5-aminolevulinate dehydratase was investigated by extended X-ray absorption fine structure (EXAFS) associated with the zinc K-edge. The enzyme binds 8 mol of zinc/mol of octameric protein, but only four zinc ions seem sufficient for full activity. We have undertaken a study on four forms of the enzyme: (a) the eight-zinc native enzyme; (b) the enzyme with only the four zinc sites necessary for full activation occupied; (c) the enzyme with the vacant sites of (b) occupied by four lead ions; (d) the product complex between (b) and porphobilinogen. We have shown that two structurally distinct types of zinc sites are available in the enzyme. The site necessary for activity has an average zinc environment best described by two/three histidines and one/zero oxygen from a group such as tyrosine or a solvent molecule at 2.06 +/- 0.02 A, one tyrosine or aspartate at 1.91 +/- 0.03 A, and one cysteine sulfur at 2.32 +/- 0.03 A with a total coordination of five ligands. The unoccupied site in (b), obtained by taking the difference spectrum between the spectra from samples (a) and (b), is dominated by a single contribution of four cysteinyl sulfur atoms at 2.28 +/- 0.02 A. Spectra from samples (c) and (d) show only small changes from that of (b), reflecting a slight rearrangement of the ligands around the zinc atom.     

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc–sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC₅₀ value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.