TY - JOUR
T1 - Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis
AU - Diefenbach, Russell J.
AU - Candy, Judith M.
AU - Mattick, John S.
AU - Duggleby, Ronald G.
PY - 1992/1/13
Y1 - 1992/1/13
N2 - A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 μM for thiamin diphosphate and from 4.21 to 45 μM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding, cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine of glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.
AB - A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 μM for thiamin diphosphate and from 4.21 to 45 μM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding, cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine of glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.
KW - Pyruvate decarboxylase
KW - Site-directed mutagenesis
KW - Thiamin diphosphate binding
KW - Zymomonas mobilis
UR - http://www.scopus.com/inward/record.url?scp=0026598760&partnerID=8YFLogxK
U2 - 10.1016/0014-5793(92)80411-9
DO - 10.1016/0014-5793(92)80411-9
M3 - Article
C2 - 1730299
AN - SCOPUS:0026598760
SN - 0014-5793
VL - 296
SP - 95
EP - 98
JO - FEBS Letters
JF - FEBS Letters
IS - 1
ER -