J. Kozarich - Academia.edu (original) (raw)

Papers by J. Kozarich

Research paper thumbnail of A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase activities

The Journal of biological chemistry, Jan 25, 1978

Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,0... more Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Research paper thumbnail of Characterization of two crystal forms of 3-carboxy- cis , cis -muconate lactonizing enzyme from Pseudomonas putida

Acta Crystallographica Section D Biological Crystallography, 1997

Two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida have b... more Two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida have been characterized. Form A is in space group P6, with unit-cell dimensions a=b=232, c=79A, ~=fl=90, 7=120°. Form B is orthorhombic, with cell dimensions a=163, b=139, c=90~,, ~=fl=7=90°.

Research paper thumbnail of Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase

Structure, 1999

Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by con... more Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Research paper thumbnail of Blood-brain barrier penetration and in vivo activity of an NGF conjugate

Blood-brain barrier penetration and in vivo activity of an NGF conjugate

Science, 1993

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and cen... more Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

Research paper thumbnail of A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase activities

The Journal of biological chemistry, Jan 25, 1978

Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,0... more Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Research paper thumbnail of Characterization of two crystal forms of 3-carboxy- cis , cis -muconate lactonizing enzyme from Pseudomonas putida

Acta Crystallographica Section D Biological Crystallography, 1997

Two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida have b... more Two crystal forms of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida have been characterized. Form A is in space group P6, with unit-cell dimensions a=b=232, c=79A, ~=fl=90, 7=120°. Form B is orthorhombic, with cell dimensions a=163, b=139, c=90~,, ~=fl=7=90°.

Research paper thumbnail of Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase

Structure, 1999

Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by con... more Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Research paper thumbnail of Blood-brain barrier penetration and in vivo activity of an NGF conjugate

Blood-brain barrier penetration and in vivo activity of an NGF conjugate

Science, 1993

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and cen... more Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.