Cyanate has been a useful reagent for studying the mechanism of a number of enzymes. The specific reaction of cyanate with hemoglobin has led to development of an automated extracorporeal system for testing the efficacy of cyanate as a drug for treatment of sickle cell anemia. Recent studies indicate that cyanate can act as a selective inhibitor of protein synthesis in animal tumors. Cyanate administered as a drug or originating naturally from spontaneous breakdown of urea (present at high levels in uremia or chronic diarrhea) has undesirable pharmacological effects. Cyanase is an inducible enzyme present in Escherichia coli which catalyzes the hydrolysis of cyanate. Unusual features of the purified cyanase from E. coli are a requirement for bicarbonate and a stable quaternary structure of 8-10 identical subunits (Mr = 16,350) where pairs of subunits may be covalently joined by disulfide bonds. The catalytic mechanism, biological role, and nature of induction by cyanate of cyanase are unknown. The availability of well characterized cyanase will be of importance if cyanase is to become a useful reagent for therapeutic or analytical purposes. The overall objectives of the proposed study are to: 1) establish the nature of the catalytic mechanism and subunit structure of cyanase (the role of bicarbonate is particularly important, since recent studies suggest that a requirement for bicarbonate as a form of metabolic regulation may be quite common); 2) isolate and characterize a mutant in the cyanase gene, and to use the cyanase deficient mutant (along with growth studies and glutathine and transcarbamoylase deficient mutants) to establish the biological role of cyanase (e.g., does cyanase function to detoxify cyanate originating from carbamoyl phosphate?) and to obtain information about cyanate metabolism and regulation of the cyanase gene (which may be unique); 3) to clone the gene for cyanase; 4) to assess the properties of an immobilized form of cyanase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033842-02
Application #
3283945
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-09-12
Project End
1987-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Kozliak, E I; Fuchs, J A; Guilloton, M B et al. (1995) Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate. J Bacteriol 177:3213-9
Anderson, P M; Korte, J J; Holcomb, T A (1994) Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate. Biochemistry 33:14121-5
Kozliak, E I; Guilloton, M B; Gerami-Nejad, M et al. (1994) Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase. J Bacteriol 176:5711-7
Anderson, P M; Korte, J J; Holcomb, T A et al. (1994) Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase. J Biol Chem 269:15036-45
Lamblin, A F; Fuchs, J A (1994) Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation. J Bacteriol 176:6613-22
Lamblin, A F; Fuchs, J A (1993) Expression and purification of the cynR regulatory gene product: CynR is a DNA-binding protein. J Bacteriol 175:7990-9
Guilloton, M B; Lamblin, A F; Kozliak, E I et al. (1993) A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli. J Bacteriol 175:1443-51
Sung, Y C; Fuchs, J A (1992) The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family. J Bacteriol 174:3645-50
Guilloton, M B; Korte, J J; Lamblin, A F et al. (1992) Carbonic anhydrase in Escherichia coli. A product of the cyn operon. J Biol Chem 267:3731-4

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