Abstract

The research plan is to explore the structure and function of E. coli alkaline phosphatase via multinuclear NMR. 31P-NMR will be used to follow formation and breakdown of the various phosphoenzyme intermediates; 113Cd-NMR to observe changes in the chemical environment of the central metal ions as a function of phosphorylation, Mg ions activation, and inhibitor binding; and 13C-NMR of enzyme biosynthetically labeled with 13C amino acids enriched in specific carbons to identify specific active site residues and metal binding residues. Bone alkaline phosphatase, specifically located in the matrix vesicle calcification system will be examined by physicochemical and enzymatic techniques as well as the 31P-NMR methods applicable to whole cells. The role of Zn(II) and Mg(II) in T7 RNA polymerase will be investigated.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (NIADDK)
Type
Research Project (R01)
Project #
5R01AM009070-21
Application #
3150750
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1975-12-01
Project End
1986-11-30
Budget Start
1984-12-01
Budget End
1986-11-30
Support Year
21
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code

  Publications

Giedroc, D P; Keating, K M; Williams, K R et al. (1987) The function of zinc in gene 32 protein from T4. Biochemistry 26:5251-9
Giedroc, D P; Coleman, J E (1986) Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase. Biochemistry 25:4969-78
Coleman, J E; Williams, K R; King, G C et al. (1986) Protein chemistry-nuclear magnetic resonance approach to mapping functional domains in single-stranded DNA binding proteins. J Cell Biochem 32:305-26
Giedroc, D P; Keating, K M; Williams, K R et al. (1986) Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein. Proc Natl Acad Sci U S A 83:8452-6
King, G C; Martin, C T; Pham, T T et al. (1986) Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347. Biochemistry 25:36-40
Giedroc, D P; Keating, K M; Martin, C T et al. (1986) Zinc metalloproteins involved in replication and transcription. J Inorg Biochem 28:155-69
Sowadski, J M; Handschumacher, M D; Murthy, H M et al. (1985) Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution. J Mol Biol 186:417-33
Gettins, P; Metzler, M; Coleman, J E (1985) Alkaline phosphatase. 31P NMR probes of the mechanism. J Biol Chem 260:2875-83