Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.
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.
Callens
M
M
Laboratorium voor Biochemie, Rijksuniversiteit Gent, Belgium.
Tomme
P
P
Kersters-Hilderson
H
H
Cornelis
R
R
Vangrysperre
W
W
De Bruyne
C K
CK
D019747:Aldose-Ketose Isomerases; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D009282:Naphthalenesulfonates; D009603:Nitroso Compounds; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology*
D002413:Cations, Divalent; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D009603:Nitroso Compounds; C001259:Fremy's salt; D003035:Cobalt; C004200:1-nitroso-3,6-disulfonate; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D002118:Calcium