Abstract
The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- De Boeck H., Loontiens F. G., De Bruyne C. K. Continuous titration of difference absorption upon binding of 4-methylumbelliferyl glycosides to concanavalin A and peanut agglutinin. Anal Biochem. 1982 Aug;124(2):308–313. doi: 10.1016/0003-2697(82)90044-6. [DOI] [PubMed] [Google Scholar]
- Schray K. J., Mildvan A. S. Kinetic and magnetic resonance studies of the mechanism of D-xylose isomerase. I. Binary and ternary complexes with manganese(II), substrates, and inhibitors. J Biol Chem. 1972 Apr 10;247(7):2034–2037. [PubMed] [Google Scholar]
- YANKEELOV J. A., Jr SPLIT-COMPARTMENT MIXING CELLS FOR DIFFERENCE SPECTROSCOPY. Anal Biochem. 1963 Sep;6:287–289. doi: 10.1016/0003-2697(63)90138-6. [DOI] [PubMed] [Google Scholar]
- Young J. M., Schray K. J., Mildvan A. S. Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions. J Biol Chem. 1975 Dec 10;250(23):9021–9027. [PubMed] [Google Scholar]