Lysyl oxidase and rrg messenger RNA.
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Animals↗
Biomedical subjects
H M Kagan
Publications and source records attributed to H M Kagan.
Specific detection of quinoproteins by redox-cycling staining.
Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.
Animals↗
Properties and function of lysyl oxidase.
Lysyl oxidase catalyzes the oxidation of peptidyl lysine to alpha-aminoadipic-delta-semialdehyde, the precursor to the covalent crosslinkages that stabilize fibers of elastin and collagen. This enzyme contains both copper and a carbonyl cofactor consistent with an o-quinone. The proposed mechanism of action is derived from available kinetic and chemical data and also can account for mechanism-based inhibition of the enzyme by specific monoamines and diamines. Recent evidence for biosynthetic precursors and for the regulation of lysyl oxidase in fibrotic and malignant diseases is discussed.
Animals↗
Induction of lung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme.
Inspiration of CdCl2 results in a focally fibrotic response in rat lungs and markedly increases the activity of lung lysyl oxidase. Western blot analyses of urea-extractable rat lung proteins revealed that the levels of an immunoreactive, 32,000-Da protein were markedly increased in the cadmium-exposed rat lung tissue, consistent with the induction of lysyl oxidase protein. Anion exchange chromatography revealed low levels of multiple peaks of catalytically functional lysyl oxidase in control rat lung extracts, while the profile of cadmium-exposed rat lung extracts displayed markedly elevated levels of multiple peaks of enzyme activity indicating that the charge heterogeneity is expressed in the activated enzyme. The cadmium-induced enzyme was purified as a species of 32 kDa, without resolving individual ionic variants. The catalytic and physical properties of the isolated enzyme were very similar to those of previously well characterized basal enzyme of bovine aorta, including the presence of a pyrroloquinoline quinone-like carbonyl cofactor. The copper and cadmium content of the cadmium-induced enzyme indicated little if any replacement of tightly-bound copper by cadmium in the exposed lung.
Aminopropionitrile↗
Structural and catalytic properties of copper in lysyl oxidase.
The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.
2,2'-Dipyridyl↗
Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.
Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
Effect of dietary cadmium and/or copper on the bone lysyl oxidase in copper-deficient rats relative to the metabolism of copper in the bone.
Effects of cadmium (Cd) on lysyl oxidase activity and copper (Cu) metabolism in bone were studied using Cu-deficient rats supplemented with Cu and/or Cd in a diet. When fed for 8 weeks on a diet containing 0.3 ppm or less Cu (-Cu diet), weanling rats revealed anemia, and markedly decreased plasma ceruloplasmin activity and serum Cu to less than 15% of normal level, showing features of Cu-deficiency. These rats were divided into four groups and refed for another 2 weeks on the following diets: Group I, -Cu diet; Group II, -Cu diet with 50 ppm Cd (+Cd diet); Group III, -Cu diet supplemented with 15 ppm Cu (+Cu diet); group IV, -Cu diet with both Cu and Cd (+Cu/+Cd diet). After 2 weeks, serum Cu levels of Groups I, II, III and IV were 1.8, 0.8, 78 and 74% of the normal control level (1.438 +/- 0.060 micrograms/ml), respectively. Concentrations of Cu in epi- and metaphyses of the control group, Groups I, II, III and IV were 1.45 +/- 0.20, 0.67 +/- 0.08, 0.76 +/- 0.12, 1.40 +/- 0.31 and 1.22 +/- 0.05 micrograms/g wet tissue, in that order. Concentrations of Cd in epi- and metaphysis increased in only Groups II and IV and were 0.15 +/- 0.03 and 0.18 +/- 0.01 micrograms/g wet tissue, respectively. Thus, having both Cd and Cu supplements in a diet did not inhibit each other's uptake into the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
Vicinal diamines as pyrroloquinoline quinone-directed irreversible inhibitors of lysyl oxidase.
The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.
Amino Acid Oxidoreductases↗
PQQ, the elusive coenzyme.
The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.
Animals↗
Comparative sensitivities of purified preparations of lysyl oxidase and other amine oxidases to active site-directed enzyme inhibitors.
Recent evidence has revealed that lysyl oxidase, plasma amine oxidase and diamine oxidase each contain copper and pyrroloquinoline quinone at their active sites as cofactors essential to their catalytic functions. It thus seems likely that these enzymes will share similar mechanisms of action. Since mechanism-based inhibitors of lysyl oxidase have important chemotherapeutic potential for the control of fibrotic disease, the relative inhibitory potential of such agents toward catalytically similar amine oxidases was assessed in the present study using purified preparations of lysyl oxidase, diamine oxidase, plasma amine oxidase and the flavin-dependent mitochondrial monoamine oxidase A and B. The results indicate that there is sufficient difference between the sensitivities of lysyl oxidase and the other amine oxidases to beta-aminopropionitrile to warrant its consideration as an antifibrotic agent in vivo, while also revealing that aminoguanidine, clorgyline and deprenyl are sufficiently selective for diamine oxidase, monoamine oxidase A and monoamide oxidase B, respectively, to differentiate between lysyl oxidase and these enzymes at appropriate concentrations.
Amine Oxidase (Copper-Containing)↗
Evidence for a functional role for histidine in lysyl oxidase catalysis.
The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.
Algorithms↗
Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase.
The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.
Amino Acid Oxidoreductases↗
Electronegativity of aromatic amines as a basis for the development of ground state inhibitors of lysyl oxidase.
Benzylamine derivatives containing para substituents of differing electronegativity as well as isomers of aminomethylpyridine have been assessed for their substrate and inhibitor potentials toward lysyl oxidase. Substituted benzylamines with increasingly electronegative para substituents had the lowest KI values and thus were the most effective inhibitors of the oxidation of elastin by lysyl oxidase. The kcat values for these compounds as substrates of lysyl oxidase were also reduced with increasingly electronegative para substituents. Both the Dkcat and D(kcat/Km) kinetic isotope effects decreased with increasingly electronegative p-substituents in [alpha, alpha'-2H]benzylamines. In contrast, there was no Dkcat solvent isotope effect with [2H] H2O while the D(kcat/Km) solvent isotope effect tended to increase with increasingly electronegative p-substituents. These results are consistent with the stabilization of an enzyme-generated substrate carbanion and thus the retardation of substrate oxidation by electronegative substituents. Such ground state stabilization thus can result in compounds with increased potential for the inhibition of the oxidation of protein substrates of lysyl oxidase.
Amino Acid Oxidoreductases↗
Alpha-proton abstraction and carbanion formation in the mechanism of action of lysyl oxidase.
Tetranitromethane (TNM) was employed as an electrophilic reagent to probe for the lysyl oxidase-catalyzed processing of n-butylamine to an intermediate carbanion during the oxidation of this amine to n-butyraldehyde according to a prior description of the use of TNM to trap enzyme-generated carbanion intermediates (Christen, P. and Riordan, J. F. (1968) Biochemistry 7, 1531-1538). The addition of n-butylamine to assay mixtures containing lysyl oxidase and TNM markedly increased the background rate of nitroform release. The Km for n-butylamine was essentially the same whether determined from the rate of lysyl oxidase-catalyzed nitroform release or from the rate of n-butyraldehyde production in the absence of TNM, the latter assessed by measurements of the rate of H2O2 formation. The initial rate of substrate- and enzyme-dependent nitroform production was linearly related to functional active site content. These data are consistent with the enzyme-dependent abstraction of an alpha-proton from the substrate to form an intermediate enzyme-bound carbanion. Kinetic analyses of the oxidation of n-butylamine and 1,1-dideutero-n-butylamine by lysyl oxidase revealed kinetic isotope effects on Vmax and Vmax/Km parameters, consistent with a rate-contributing alpha-proton abstraction step. These and other available data are incorporated into a proposal for the mechanism of action of this enzyme.
Amino Acid Oxidoreductases↗
Stimulation of lysyl oxidase (EC 1.4.3.13) activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth-muscle cells.
Previous studies have indicated a greater incidence of atherosclerotic cardiovascular disease in men than in women of child-bearing age, suggesting that vascular interactions with sex steroids may effect pathogenesis in these cases. In the present study, it was found that the presence of 10-100 nM-testosterone in the growth medium of calf aortic smooth-muscle cells in culture stimulates lysyl oxidase activity approx. 2.5-fold in the medium and 5.5-fold in the fraction bound to the cell layer. Androgen receptors were identified in these cultured smooth-muscle cells, and their properties were very similar to those in the cytosolic fraction of whole bovine aortic tissue. These receptors appeared to be specific for androgen, of high affinity (Kd = 0.4 nM) and of low capacity (9000 sites/cell). The present results indicate that the aortic smooth-muscle cell is a cellular target for androgens, and thus raise the possibility that the development of fibrotic arterial lesions involving the deposition of excess collagen may in part be regulated by androgen-mediated stimulation of collagen cross-linkage formation as catalysed by lysyl oxidase.
Amino Acid Oxidoreductases↗
Evidence for pyrroloquinolinequinone as the carbonyl cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy.
The present study investigated the possibility that pyrroloquinolinequinone (PQQ), an aromatic carbonyl recently indicated to be the carbonyl cofactor in bovine plasma amine oxidase, may also be present at the active site of lysyl oxidase. The absorption and resonance Raman spectra of the phenylhydrazones of bovine plasma amine oxidase, of peptides derived from the active site of bovine aorta lysyl oxidase, and of PQQ were very similar, indicating that the carbonyl cofactor of lysyl oxidase is PQQ or a compound which closely resembles PQQ.
Amino Acid Oxidoreductases↗
Reaction pathway of bovine aortic lysyl oxidase.
The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.
Aldehydes↗
Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate.
Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.
Amino Acid Oxidoreductases↗