Abstract

We have used site-directed spin labeling to prove the active site of phosphoribulokinase, an enzyme in the Calvin cycle that catalyzes the transfer of a phosphoryl group from ATP to ribulose 5-phosohate. To this end, we have generated two single cysteine mutants A16C and k53C by site directed mutagenesis, in the active site of PRK. The two mutants were characterized for activity and also checked for structural perturbation using filtration, CD and titration of the active site with the spin labeled ATP probe, ATPSAP. The mutants show no noticeable deviation from Wild Type. They were then spin labeled with MTSSL and their spectra recorded in the presence and absence of ATP and the allosteric effector NADH. SL-K53C spectrum was not sensitive to ATP or NADH while the A16C spectrum was sensitive to ATP. The latter is expected due to the fact that A16 is a residue in the P-loop which is known to be the nucleotide binding site. Experiments are in progress on the double mutant A16C/K53C to determine distances in the active site. ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001008-25
Application #
6307890
Study Section
Project Start
2000-03-01
Project End
2001-02-28
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
25
Fiscal Year
2000
Total Cost
$11,274
Indirect Cost

  Institution

Name
Medical College of Wisconsin
Department
Type
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226

  Publications

Mao, Li; Liu, Yu-Xiang; Huang, Chun-Hua et al. (2015) Intrinsic Chemiluminescence Generation during Advanced Oxidation of Persistent Halogenated Aromatic Carcinogens. Environ Sci Technol 49:7940-7
Shan, Guo-Qiang; Yu, Ao; Zhao, Chuan-Fang et al. (2015) A combined experimental and computational investigation on the unusual molecular mechanism of the Lossen rearrangement reaction activated by carcinogenic halogenated quinones. J Org Chem 80:180-9
Li, Yan; Huang, Chun-Hua; Liu, Yu-Xiang et al. (2014) Detoxifying polyhalogenated catechols through a copper-chelating agent by forming stable and redox-inactive hydrogen-bonded complexes with an unusual perpendicular structure. Chemistry 20:13028-33
Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang et al. (2013) Ofloxacin induces apoptosis via ?1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes. Toxicol Appl Pharmacol 267:74-87
Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang et al. (2013) Bisphenol A at a low concentration boosts mouse spermatogonial cell proliferation by inducing the G protein-coupled receptor 30 expression. Toxicol Appl Pharmacol 267:88-94
Shao, Jie; Huang, Chun-Hua; Kalyanaraman, Balaraman et al. (2013) Potent methyl oxidation of 5-methyl-2'-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism. Free Radic Biol Med 60:177-82
Sheng, Zhi-Guo; Li, Yan; Fan, Rui-Mei et al. (2013) Lethal synergism between organic and inorganic wood preservatives via formation of an unusual lipophilic ternary complex. Toxicol Appl Pharmacol 266:335-44
Qin, Hao; Huang, Chun-Hua; Mao, Li et al. (2013) Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens. Free Radic Biol Med 63:459-66
Huang, Chun-Hua; Shan, Guo-Qiang; Mao, Li et al. (2013) The first purification and unequivocal characterization of the radical form of the carbon-centered quinone ketoxy radical adduct. Chem Commun (Camb) 49:6436-8
Liddle, Brendan J; Wanniarachchi, Sarath; Hewage, Jeewantha S et al. (2012) Electronic communication across diamagnetic metal bridges: a homoleptic gallium(III) complex of a redox-active diarylamido-based ligand and its oxidized derivatives. Inorg Chem 51:12720-8

Showing the most recent 10 out of 368 publications