Gram Atoms Of Zinc Research Articles

The catalytic mechanism of hamster dihydroorotase.

Dihydroorotase catalyses the third reaction of the pathway for de novo biosynthesis of pyrimidine nucleotides. The reaction catalysed involves an intramolecular ring closure of N-carbamyl-L-aspartate (CA-asp) to form L-dihydroorotate (DHO), a dihydropyrimidine. The interconversion of CA-asp and DHO is freely reversible (Christopherson and Jones, 1979) and the pH-rate profiles for the biosynthetic and degradative reactions and inhibition of the enzyme by L-cysteine suggested that mammalian dihydroorotase has a catalytic mechanism similar to carboxypeptidase A (Christopherson and Jones, 1980) with a zinc atom at the active site. This zinc atom would polarise the =C=O group of the scissile peptide bond in the dihydropyrimidine ring of DHO and stabilise the tetrahedral, dioxy anionic transition state of the reaction (Fig. 1). Taylor et al. (1976) had purified the dihydroorotase from Clostridium oroticum and found two gram atoms of zinc per subunit and similar pH-rate profiles. Kelly et al. (1986) subsequently demonstrated that hamster dihydroorotase also contained an atom of zinc which was required for catalysis. It therefore seems likely that mammalian dihydroorotase does have a catalytic mechanism which resembles that of zinc protease.

An improved method for isolation and identification of Zn-metallothionein from cadmium-induced rat liver.

Zn-metallothioneins (MT-1 and MT-2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G-50 and DEAE Sephadex A-50, new chromatographic media produced by Pharmacia, Sephacryl S-200, S-100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT-1 and MT-2 contained 6 gram atoms of zinc, but no cadmium. Purified MT-1 and MT-2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd-MT.

Formaldehyde dehydrogenase from Pseudomonas putida: a zinc metalloenzyme.

The NAD+-dependent formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 4 gram atoms of zinc per mol, corresponding to 2 gram atoms of zinc per subunit monomer. Treatment of the enzyme with o-phenanthroline resulted in removal of 1 gram atom of zinc per subunit and caused a complete inactivation of the enzyme. The activity lost was restored by the addition of zinc ions, by which the zinc content was also reversed to almost the same level as that of the native enzyme. Another zinc atom that was resistant to metal chelator-treatment was liberated from the enzyme only after the irreversible denaturation of the enzyme. These results indicate that the formaldehyde dehydrogenase of P. putida is a zinc metalloenzyme and one of two zinc atoms per subunit participates in the catalytic activity of the enzyme, another zinc being presumably involved in maintaining the native conformation of the enzyme. Treatment of the enzyme with bipyridine also caused a reversible inactivation of the enzyme, but the zinc content remained unchanged. The spectrophotometric analysis indicated that the formation of a enzyme-Zn-bipyridine complex took place. Incubation of the enzyme with p-chloromercuribenzoate also resulted in a complete loss of the activity. These results suggest that an intrinsic zinc and sulfhydryl group together with NAD+ participate in the dehydrogenation reaction of substrate by the enzyme.

Purification and characterization of animal porphobilinogen synthases—II. Dog liver porphobilinogen synthase

1. 1. A method is described for the purification of porphobilinogen synthase (PBG-S) from dog liver (acetone dry powder; ammonium sulfate fractionation: controlled heat denaturation; ion exchange chromatography; gel filtration). The 911-fold purified enzyme with a specific activity of 372 in U/mg and 6.2 nkat/mg enzyme protein, respectively, appears as a single band in disc electrophoresis. 2. 2. The optimum pH of the enzyme is 6.8; the Michaelis constant K m = 1.77 · 10 −4M with 5-aminolevulinic acid as substrate. 3. 3. The determination of the molecular weight of the native multisubunit enzyme (268,000 dalton) and of the subunit (33,500 dalton) are in favour of an octameric structure. 4. 4. The acyi group blocking the N-terminus has been identified as an acetyl group (hydrazinolysis; dansylation;TLC: HPLC). 5. 5. The amino acid composition of the PBG-S subunit is as follows CH 3CO-NH-(Asx 22Thr 9–10Ser 18Glx 28–29Pro 20–21Gly 22Ala 35–37Val 23–25Met 5 Ile 8–9Leu 32–33Tyr 10Phe 12Lys 12Cys 3HiS 7Arg 22Trp 3)-Ala-Leu-COOH. 6. 6. In the presence of ganidinium chloride only one free sulfhydryl group per subunit (3 cysteine residues) could be detected. 7. 7. The purified enzyme contains 0.56 gram atoms of zinc per octamer (neutron activating analysis).

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.

Human pancreatic carboxypeptidase B. I. Isolation, purification, and characterization of fraction II

Human carboxypeptidase B fraction II has been purified from pancreatic juice by DEAE-‘Sephadex’ chromatography, isoelectric focusing, and ‘Sephadex’ G-100 gel filtration. The enzyme has been characterized by analytical polyacrylamide disc-gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, K m determination, molecular weight determination on ‘Sephadex’ G-100, zinc analysis, and inhibition by metal chelating agents. Human carboxypeptidase B fraction II appeared homogeneous in analytical polyacrylamide disc-gel electrophoresis, but showed two components of 23,500 and 9,200 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Zinc analysis revealed 0.96 gram atoms of zinc per mole of enzyme, and a K m of 65 ± 3 μM was determined for hydrolysis of hippuryl- l-arginine.

Studies on a nuclease from Penicillium citrinum. V. Some physical and chemical properties of nuclease P1.

Some physico-chemical properties of nuclease P1 (isoelectric point, 4.5) from Penicillium citrinum were studied. The extinction coefficient at 280 nm, , was 18.4. The partial specific volume , the intrinsic viscosity ([η]), the sedimentation coefficient () and the diffusion coefficient (D20,w) were 0.712 ml/g, 4.17 × 10−2 ml/g, 3.55 S and 7.4 × l0−7 cm2/sec, respectively.The molecular weight was estimated to be 42,000~50,000 by several methods including sedimentation velocity, sedimentation equilibrium, gel filtration and SDS-acrylamide gel electrophoresis. The amino acid composition of the enzyme was characterized by the high content of hydrophobic amino acids, especially tyrosine and tryptophan. The enzyme was found to be a zinc metalloenzyme which contained 3 gram atoms of zinc per mole based upon the molecular weight of 44,000. The enzyme also contained about 17.4% carbohydrates, consisting of mannose, galactose and glucosamine in a molar ratio of 6: 2: 1. The enzyme exhibited a high affinity for conc...

Bovine erythrocyte cupro-zinc protein. 2. Physicochemical properties and circular dichroism.

The physicochemical properties and circular dichroism of bovine erythrocyte cupro‐zinc protein were investigated. In sedimentation velocity experiments in the ultacentrifuge, the protein sedimented as a single peak with an s°20,w of 3.06 S. The diffusion coefficient was 8.27 F. the partial specific volume 0.736 ml/g, and the instrinsic viscosity 0.033 (g/100 ml)‐1. Combination of these hydrodynamic data indicated a molecular weight near 33 000, which was in good agreement with chemical and gel filtration data. The molecular weight was also determined by the sedimentation equilibrium method using interference optics. By this method M̄w was 33 500, and the protein appeared to be homogeneous. The circular dichroism spectrum of bovine erythrocyte cupro‐zinc protein was unique among copper proteins in having positive contributions at 610, 440 and 350 nm, which disappeared on removing the metal atoms. In the ultraviolet there were positive contributions near 260, 270 and 290 nm, and removal of the metal atoms changed these into negative contributions at 265 and 285 nm and a positive contribution at 295 nm. Possible assignments of these bands are discussedd. In the far ultraviolet, the circular dichroism spectra of the native protein and apoprotein showed few differences, indicating that the metal atoms do not play a dominant role in determining protein conformation. However, urea had a profound effect on the circular dichroism of the apoprotein but little effect on that of the native protein. Neither the native protein nor the apoprotein showed the duble minimum between 200 and 220 nm characteristic of α‐helix, but rather the existence of a minimum at 210 nm suggested that the proteins contained some β‐pleated sheet structure. Reduction and carboxymethylation of thiol groups greatly reduced the ultraviolet circular dichroism, indicating that the disulphide bridges have a strong influence in determining the tertiary structure of the protein.

Carboxypeptidase A: approaches to the chemical nature of the active center and the mechanisms of action.

in 0.02 M sodium Veroral-2 M NaCI, pH 7.5 buffer at 0? for a reaction time of thirty min. The pH of the reaction was maintained at 7.5 by the addition of 1 M NaOH by means of a pH Stat. Excess anhydride was removed by dialysis against the same buffer at 0? for 48 hr. [odinations were performed with a 35-fold molar excess of 12 in 0.5 M KI at 25? for ten min. Photooxidations were carried out in the presence of methylene blue according to the method of Weil and Buchert.7 All chemicals used in these experiments were reagent grade and used without further purification except for acetic anhydride which was redistilled. Results and Discussion.-The metal binding site of apocarboxypeptidase: Ag+, p-mercuribenzoate, or ferricyanide titrate only one -SH group in the apocarboxypeptidase and none in the metallocarboxypeptidasel2 (Table 1). A peptide containing the single cysteine residue of carboxypeptidase to which zinc is bound has recently been isolated. The sequence and magnitude of the stability constants of different metallocarboxypeptidases, their comparison to those of simple ligands and complexometric titrations indicated early that the metal atom is also bound to nitrogen.' 12 The experiments suggesting that the a-amino group of the N-terminal asparagine (AspNH2) (Table 1) may be the second donor group for zinc binding are quite analogous to those employed for the -SH group. On exposure to fluorodinitrobenzene or phenylisothiocyanate, the apoenzymel readily forms DNP-aspartic acid (DNP-Asp) or PTH-asparagine (PTN-AspNH2), respectively, while the yields from the metalloenzyme are significantly less. Zinc appears to be displaced from the enzyme in proportion to the reaction of these reagents with the N-terminal asparagine. The sum of the number of gram atoms of zinc bound per mole of