Progress 10/01/02 to 09/30/03
Outputs
Progress Report for 2002-2003 The protocol for the isolation of arginine kinase from the cockroach, results in 6.6 mg of pure enzyme from 6.8g of whole cockroach. Cockroach arginine kinase has a molecular mass approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50oC for 10 min) and has a pH optima of 8.5 and 6.5 to 7.5 for the forward and reverse reactions, respectively. Treatment with dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and interestingly, the reaction is biphasic. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha helical structure. The intrinsic protein fluorescence emission maximum at 340 nm, suggests that tryptophan
residues are below the surface of the protein and not exposed to solvent. The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach was investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for motility. Analysis reveals a series of dissociation constants (in mM) for the binary complex and the ternary complex. For each substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating insignificant binding cooperativity. Progress curves demonstrate formation of an inhibitory catalytic dead-end complex. The inhibitory effect and judged by a decrease in initial velocity is not enhanced by acetate and moderately enhanced by halides, borate and SCN-,. Nitrite and nitrate decrease the initial velocity by 75 and 95%, respectively. However, the while actual percent inhibition after nine minutes of assay is
also greatest for nitrate and nitrite, we observe 51% inhibition with borate. Determination of the ratio of the initial velocity to the degree of overall inhibition results in factor which we propose demonstrates that the profound inhibition by nitrate is mechanistically more complex than stabilization of the dead-end complex, while borate is the most effective in formation of a transition state analog. Additional kinetic analysis shows that nitrate anion is a non-competitive inhibitor (Ki=8.0 mM) with respect to arginine. While not a substrate for arginine kinase from the cockroach, D-arginine is an effective competitive inhibitor with Ki = 0.31 mM. L-Canavanine is a poor substrate arginine kinase with a Km = 6.7 and a Vmax with the pure enzyme approximately one-sixth that of L-arginine. Factors such as L-arginine being an essential amino acid for the cockroach, D-arginine and canavanine being toxic to insects and the inhibitory effect of nitrate in stabilizing a dead-end all suggest
that a cocktail of effector molecules for arginine kinase, an enzyme required for motility, could function to control infestations.
Impacts
Our studies have defined several physical and catalytic features of the pure arginine kinase from the cockroach. Application of this information to designing and testing inhibitors of this enzyme, essential for mobility will lead to (a) a better understanidng of the physiology and biochemistry of cockroach bioenergics and metabolism (b) a rational approach to inhibition of activity with potential control of cockroach popultion. In addition to the cockroach being a serious destructive agent in agriculture, the cockroach enzyme arginine kinase is a potent immunogen in humans responsible, in part, for asthma in populations residing in areas of high infestation.
Publications
- Brown, A. E., France, R. M. and Grossman, S. H. (2004) Purification and Characterization of Arginine Kinase from the American Cockroach (Periplaneta americana). Archives of Insect Biochemistry and Physiology in press
- Brown, Ashli and Grossman, Steven (abstract) Kinetics and inhibition of Arginine Kinase from the American Cockroach (Periplaneta americana). SERMACS, Annual Meeting, American Chemical Society, Atlanta, 2003 p.91
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Progress 10/01/00 to 09/30/01
Outputs
We have developed a rapid and high yield purification of arginine kinase from the American cockroach. The enzyme is a monomer of molecular weight 43.5 kDal. The enzyme is thermostable up to 68 degree C, exhibits a pH optimum of 8.5 and has one highly reactive sulfhydryl. Kinetic analysis for the phosphotransferase reaction in both directions reveals that the nucleotide substrate binds with a lower dissociation constant compared to the phosphagen substrate. Analysis comparing the binary comples with ternary complex in both directions, however, indicates little cooperativity in substrate binding. The observation that monovalent cations are inhibitory, suggest that as with other phosphagen kinases, arginine kinase from the cockroach forms a highly stable dead-end complex. Formation of this complex may form the basis for developing agents which could act to inhibit energy transduction in the cockroach.
Impacts
The isolation and physical and kinetic characterization has provided evidence for development of substrate analogs and transition state analogs which would be useful as potential poisons. These agents then may be used in population control for the cockroach
Publications
- Brown, A. and Grossman, S. H. (2002) Purification and characterization of arginine kinase from the American Cockroach. SERMACS 2002 abstract #67, Charleston S.C (11/13/2002)
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