Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber

• Published in: Biochemical journal Vol. 250; no. 1; pp. 285 - 290
• Main Authors: Callens, M, Tomme, P, Kersters-Hilderson, H, Cornelis, R, Vangrysperre, W, De Bruyne, C K
• Format: Journal Article
• Language: English
• Published: England 15-02-1988
• Subjects: absorption spectroscopy; Aldose-Ketose Isomerases; Binding Sites; Binding, Competitive; Calcium - metabolism; Carbohydrate Epimerases - metabolism; Cations, Divalent - metabolism; cobalt; Cobalt - metabolism; Macromolecular Substances; magnesium; Magnesium - metabolism; Naphthalenesulfonates; Nitroso Compounds; Protein Conformation; Spectrophotometry, Ultraviolet; Streptomyces - enzymology; Streptomyces yiolaceoruber; xylose isomerase
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2500285

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.