共查询到20条相似文献,搜索用时 31 毫秒
1.
Orellana MS López V Uribe E Fuentes M Salas M Carvajal N 《Archives of biochemistry and biophysics》2002,403(2):155-159
Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn(2+). The effect was reversed by hydroxylamine and involved a residue with a pK(a) of 6.5+/-0.1. Half activation with Mn(2+) was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in K(m) for arginine or K(i) for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn(2+) and contained <0.1 Mn(2+)/subunit, whereas H101N was half active and contained 1.2+/-0.1 Mn(2+)/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state. 相似文献
2.
López V Alarcón R Orellana MS Enríquez P Uribe E Martínez J Carvajal N 《The FEBS journal》2005,272(17):4540-4548
To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II. 相似文献
3.
Carvajal N Salas M López V Uribe E Herrera P Cerpa J Fuentes M 《Journal of inorganic biochemistry》1999,77(3-4):163-167
Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor. 相似文献
4.
A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+. 相似文献
5.
The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant. 相似文献
6.
Assay and kinetics of arginase 总被引:1,自引:0,他引:1
A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase. 相似文献
7.
Human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was immobilised by attachment to nylon with glutaraldehyde as a crosslinking agent. Incubation of the immobilised tetrameric enzyme with EDTA followed by dialysis resulted in the dissociation of the enzyme into inactive matrix-bound and solubilised subunits. Both species recovered enzymatic activity after incubation with Mn2+, and the activity of the reactivated matrix-bound subunits was nearly 25% of that shown by the enzyme initially attached to the support in the tetrameric form. When the reactivated bound subunits were incubated with soluble subunits in the presence of Mn2+, they 'picked-up' from the solution an amount of protein and enzymatic activity almost identical to that initially lost by the immobilised tetramer after the dissociating treatment with EDTA. This occurred only in the presence of Mn2+. It is suggested that the reactivation of the subunits of arginase involves the initial formation of an active monomer, which then acquires a conformation that favours a reassociation to the tetrameric state. 相似文献
8.
Dabir S Dabir P Somvanshi B 《Journal of enzyme inhibition and medicinal chemistry》2006,21(6):727-731
The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn2+ concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55 degrees C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35 degrees C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed. 相似文献
9.
Arginase is a binuclear manganese metalloenzyme that hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of D128E, D128N, D232A, D232C, D234E, H101N, and H101E arginases I have been determined by X-ray crystallographic methods to elucidate the roles of the first-shell metal ligands in the stability and catalytic activity of the enzyme. This work represents the first structure-based dissection of the binuclear manganese cluster using site-directed mutagenesis and X-ray crystallography. Substitution of the metal ligands compromises the catalytic activity of the enzyme, either by the loss or disruption of the metal cluster or the nucleophilic metal-bridging hydroxide ion. However, the substitution of the metal ligands or the reduction of Mn(2+)(A) or Mn(2+)(B) occupancy does not compromise enzyme-substrate affinity as reflected by K(M), which remains relatively invariant across this series of arginase variants. This implicates a nonmetal binding site for substrate l-arginine in the precatalytic Michaelis complex, as proposed based on analysis of the native enzyme structure (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W. (1996) Nature 383, 554-557). 相似文献
10.
Holowatz LA Kenney WL 《American journal of physiology. Heart and circulatory physiology》2007,293(2):H1090-H1096
Full expression of reflex cutaneous vasodilation (VD) is dependent on nitric oxide (NO) and is attenuated with essential hypertension. Decreased NO-dependent VD may be due to 1) increased oxidant stress and/or 2) decreased L-arginine availability through upregulated arginase activity, potentially leading to increased superoxide production through uncoupled NO synthase (NOS). The purpose of this study was to determine the effect of antioxidant supplementation (alone and combined with arginase inhibition) on attenuated NO-dependent reflex cutaneous VD in hypertensive subjects. Nine unmedicated hypertensive [HT; mean arterial pressure (MAP) = 112 +/- 1 mmHg] and nine age-matched normotensive (NT; MAP = 81 +/- 10 mmHg) men and women were instrumented with four intradermal microdialysis (MD) fibers: control (Ringer), NOS inhibited (NOS-I; 10 mM N(G)-nitro-L-arginine), L-ascorbate supplemented (Asc; 10 mM L-ascorbate), and Asc + arginase inhibited [Asc+A-I; 10 mM L-ascorbate + 5 mM (S)-(2-boronoethyl)-L-cysteine-HCl + 5 mM N(omega)-hydroxy-nor-L-arginine]. Oral temperature was increased by 0.8 degrees C via a water-perfused suit. N(G)-nitro-L-arginine was then ultimately perfused through all MD sites to quantify the change in VD due to NO. Red blood cell flux was measured by laser-Doppler flowmetry over each skin MD site, and cutaneous vascular conductance (CVC) was calculated (CVC = flux/MAP) and normalized to maximal CVC (%CVC(max); 28 mM sodium nitroprusside + local heating to 43 degrees C). During the plateau in skin blood flow (Delta T(or) = 0.8 degrees C), cutaneous VD was attenuated in HT skin (NT: 42 +/- 4, HT: 35 +/- 3 %CVC(max); P < 0.05). Asc and Asc+A-I augmented cutaneous VD in HT (Asc: 57 +/- 5, Asc+A-I: 53 +/- 6 %CVC(max); P < 0.05 vs. control) but not in NT. %CVC(max) after NOS-I in the Asc- and Asc+A-I-treated sites was increased in HT (Asc: 41 +/- 4, Asc+A-I: 40 +/- 4, control: 29 +/- 4; P < 0.05). Compared with the control site, the change in %CVC(max) within each site after NOS-I was greater in HT (Asc: -19 +/- 4, Asc+A-I: -17 +/- 4, control: -9 +/- 2; P < 0.05) than in NT. Antioxidant supplementation alone or combined with arginase inhibition augments attenuated reflex cutaneous VD in hypertensive skin through NO- and non-NO-dependent mechanisms. 相似文献
11.
N Carvajal V López M Salas E Uribe P Herrera J Cerpa 《Biochemical and biophysical research communications》1999,258(3):808-811
Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme. 相似文献
12.
Y Satoh 《Biochimica et biophysica acta》1986,872(3):177-182
The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+. 相似文献
13.
The human malaria parasite Plasmodium falciparum possesses a single gene with high similarity to the metalloproteins arginase and agmatinase. The recombinant protein reveals strict specificity for arginine, and it has been proposed that its function in ornithine production is as a precursor for polyamine biosynthesis. The specific activity of the plasmodial arginase was found to be 31 micromol min(-1) mg(-1) protein and the k(cat) was calculated as 96 (s-1) . The Km value for arginine and Ki value for ornithine were determined as 13 mM and 19 mM, respectively. The active arginase is a homotrimer of ca. 160 kDa. Dialysis of the arginase against EDTA results in monomers of approximately 48 kDa; however, the quaternary structure can be restored by addition of Mn 2+ . Mutagenic analyses of all the amino acid residues proposed to be involved in metal binding led to complex dissociation, except for the His-193-Ala mutant, which was also inactive but retained the trimeric structure. Substitution of His-233, which has been suggested to be in charge of proton shuttling within the active site, disrupted the trimeric structure and thereby the activity of the Pf arginase. Northern blot analysis identified a stage-specific expression pattern of the plasmodial arginase in the ring/young trophozoite stage, which guarantees the provision of ornithine for essential polyamine biosynthesis. 相似文献
14.
Ensunsa JL Symons JD Lanoue L Schrader HR Keen CL 《Experimental biology and medicine (Maywood, N.J.)》2004,229(11):1143-1153
L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected. 相似文献
15.
Tolmacheva AS Zaksas NP Buneva VN Vasilenko NL Nevinsky GA 《Journal of molecular recognition : JMR》2009,22(1):26-37
It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed. 相似文献
16.
The estrone 3-sulfate 16 alpha-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O2 ratio of 0.64 caused 50% inhibition of activity. Since inhibition was also obtained in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate . HCl it seems likely that the hydroxylase is a cytochrome P450 containing system. A fourfold increase in enzyme activity was brought about by 40 mM Mg2+ or Ca2+ while the same concentration of Mn2+ resulted in a twofold increase. Lesser increases were seen with Na+ or K+ and complete inhibition was obtained in the presence of Fe2+, Cu2+, or EDTA. When assayed in the presence of detergent concentrations sufficiently small to guard against cytochrome P450 destruction, it was found that Cutscum, Triton X-100, and Triton N-101 each caused greatest inhibition of enzyme activity. Lesser inhibition was apparent in the presence of Miranol H2M, cholate, or deoxycholate. The nonionic detergent, Brij 35, caused least inhibition of all and, when hepatic microsomes were treated higher concentrations of Brij 35, about 80% of protein and over 95% cytochrome P450 were to be found in the 100 000 X g supernatant. Microsomal activity was more stable when stored at -20 degrees C in buffer containing glycerol, EDTA, and dithiothreitol than in buffer alone. Under best conditions only 10% of the hydroxylase activity was lost in one week. 相似文献
17.
Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia 总被引:1,自引:0,他引:1
In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high. In arginine-grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo. 相似文献
18.
Tormanen CD 《Journal of inorganic biochemistry》2003,93(3-4):243-246
Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase. 相似文献
19.
Crystalline L-histidine ammonia-lyase of Achromobacter liquidum was prepared with a 24% recovery of the activity. The specific activity of the pure enzyme (63 mumol of urocanic acid min-1 mg-1) is similar to those so far reported for the enzyme from other sources. The purified enzyme appeared to be homogeneous by analytical disc electrophoresis and isoelectric focusing (pI = 4.95). The molecular weight determined by Sephadex G-200 gel filtration is 200000. The optimum pH is 8.2, and the optimum temperature is 50 degrees C. The enzyme showed strict specificity to L-histidine (Km = 3.6 mM). Several histidine derivatives are not susceptible to the enzyme but do inhibit the enzyme activity competitively; the most effective inhibitors are L-histidine methyl ester (Ki = 3.66 mM) and beta-imidazole lactic acid (Ki = 3.84 mM). L-Histidine hydrazide (Ki = 36 mM) and imidazole (Ki = 6 mM) noncompetitively inhibited the enzyme EDTA markedly inhibited enzyme activity and this inhibition were reversed by divalent metal ions such as Mn2+, Co2+ Zn2+, Ni2+, Mg2+, and Ca2+. These results suggest that the presence of divalent metal ions is necessary for the catalytic activity of histidine ammonia-lyase. Sodium borohydride and hydrogen peroxide inhibited the enzyme activity. 相似文献
20.
J García-Martínez A Linares M D Suárez E García-Peregrín 《Revista Espanola de Fisiología》1982,38(3):261-266
Mevalonate kinase from neonatal chick liver has been partially purified by ammonium sulphate precipitation and Sephadex G100 and DEAE-cellulose fractionation. The kinetic characteristics agreed with the sequential mechanism suggested for the enzyme and provided apparent Km values of 0.01 mM for mevalonic acid and 0.25 mM for ATP. Partially purified mevalonate kinase from neonatal chick liver showed an absolute specificity for ATP. Mn2+ was a better activator than Mg2+ at low concentrations (0.1-1.0 mM). Higher Mn2+ concentrations produced a clear inhibition of mevalonate kinase. Likewise, addition of EDTA, with or without metal ions, clearly inhibited the enzymatic reaction. 相似文献