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Characterization and structural determination of metalloenzymes: DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-a decarbonylase/synthase

Arndt, Joseph W.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1061312369

Abstract Details

2003, Doctor of Philosophy, Ohio State University, Chemistry.
My research focused on the structure determination of proteins from three metalloenzyme systems by X-ray crystallography. The first target was rat DNA polymerase b, which catalyzes the template-directed nucleotidyl transfer reaction required for DNA replication. We have determined the crystal structures of two intermediate complexes in the reaction pathway of this enzyme, (i) a pre-chemistry ternary complex containing protein, DNA, and a chromium dNTP analog and (ii) a post-chemistry complex after nucleotide incorporation. These intermediate structures have allowed us to dissect the role of the two essential magnesium ions in initiating the enzymes conformational change. Based on these structures, a revised mechanism for replication and fidelity is proposed. The second part of this research involved structural studies on a carboxypeptidase (PfuCP) from the hyperthermophilic archaeon, Pyrococcus furiosus. Like other carboxypeptidases, it catalyzes the removal of amino acids from the C-terminus of protein and peptide chains. In this project we have solved three different structures of this enzyme, an apo form and two metal-bound forms. The overall fold of this enzyme is distinct from all other known structures of carboxypeptidase. It differs significantly in sequence, however, with one important feature being a consensus HEXXH metal-binding motif at its active site. While HEXXH motifs are common in aminopeptidases and endopeptidases, this is the first observation of this motif in a carboxypeptidase. These structures represent the first prototype available for this growing family of carboxypeptidase. Comparison with other metallopeptidases allowed us to propose a catalytic mechanism of its C-terminal peptide hydrolysis reaction. The last target of this research was the Methanosarcina barkeri acetyl coenzyme-A decarbonylase/synthase (ACDS) complex and its five individual components. The ACDS complex is involved in the metabolism acetyl coenzyme-A by methanogens. In this project we have isolated ACDS and its subunits, a2e2, b, g, and d, for crystallization and subsequent structure elucidation. Several crystal forms have been identified for the a2e2 and b components. In addition, we have performed metal chelation and reconstitution experiments on the b component that indicate the enzymes acetyl transferase activity is dependent on nickel, and not copper. My research focused on the structure determination of proteins from three metalloenzyme systems by X-ray crystallography. The first target was rat DNA polymerase b, which catalyzes the template-directed nucleotidyl transfer reaction required for DNA replication. We have determined the crystal structures of two intermediate complexes in the reaction pathway of this enzyme, (i) a pre-chemistry ternary complex containing protein, DNA, and a chromium dNTP analog and (ii) a post-chemistry complex after nucleotide incorporation. These intermediate structures have allowed us to dissect the role of the two essential magnesium ions in initiating the enzymes conformational change. Based on these structures, a revised mechanism for replication and fidelity is proposed. The second part of this research involved structural studies on a carboxypeptidase (PfuCP) from the hyperthermophilic archaeon, Pyrococcus furiosus. Like other carboxypeptidases, it catalyzes the removal of amino acids from the C-terminus of protein and peptide chains. In this project we have solved three different structures of this enzyme, an apo form and two metal-bound forms. The overall fold of this enzyme is distinct from all other known structures of carboxypeptidase. It differs significantly in sequence, however, with one important feature being a consensus HEXXH metal-binding motif at its active site. While HEXXH motifs are common in aminopeptidases and endopeptidases, this is the first observation of this motif in a carboxypeptidase. These structures represent the first prototype available for this growing family of carboxypeptidase. Comparison with other metallopeptidases allowed us to propose a catalytic mechanism of its C-terminal peptide hydrolysis reaction. The last target of this research was the Methanosarcina barkeri acetyl coenzyme-A decarbonylase/synthase (ACDS) complex and its five individual components. The ACDS complex is involved in the metabolism acetyl coenzyme-A by methanogens. In this project we have isolated ACDS and its subunits, a2e2, b, g, and d, for crystallization and subsequent structure elucidation. Several crystal forms have been identified for the a2e2 and b components. In addition, we have performed metal chelation and reconstitution experiments on the b component that indicate the enzymes acetyl transferase activity is dependent on nickel, and not copper.
Michael Chan (Advisor)
194 p.
Chemistry, Biochemistry
DNA polymerase; carboxypeptidase; metalloenzyme; crystal structure; methanogen

Recommended Citations

Citations

  • Arndt, J. W. (2003). Characterization and structural determination of metalloenzymes: DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-a decarbonylase/synthase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1061312369

    APA Style (7th edition)

  • Arndt, Joseph. Characterization and structural determination of metalloenzymes: DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-a decarbonylase/synthase. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1061312369.

    MLA Style (8th edition)

  • Arndt, Joseph. "Characterization and structural determination of metalloenzymes: DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-a decarbonylase/synthase." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1061312369

    Chicago Manual of Style (17th edition)

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