Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects.
The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.
van Bastelaere
P B
PB
Laboratory of Biochemistry, University of Ghent, Belgium.
Kersters-Hilderson
H L
HL
Lambeir
A M
AM
D000191:Actinomycetaceae / Q000201:enzymology* / Q000235:genetics; D019747:Aldose-Ketose Isomerases; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D016296:Mutagenesis; D011485:Protein Binding; D011522:Protons; D012997:Solvents; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D011522:Protons; D012997:Solvents; D014994:Xylose; D003903:Deuterium; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose