G. Schenk et al., THE ROLE OF HIS113 AND HIS114 IN PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS, European journal of biochemistry, 248(1), 1997, pp. 63-71
Pyruvate decarboxylase (PDC) is one of several enzymes that require th
iamin diphosphate (ThDP) and a divalent cation as essential cofactors.
Recently, the three-dimensional structures of the enzyme from two yea
sts have been determined. While these structures shed light on the bin
ding of the cofactors and the reaction mechanism, the interactions bet
ween the substrate pyruvate and the enzyme remain unclear. We have use
d PDC from Zymomonas mobilis as a model for these enzymes in order to
study substrate binding. The recombinant enzyme was expressed in Esche
richia coli. High yield, simplicity of purification, high stability an
d simple kinetics make this model well suited for these studies. Activ
ity measurements in the pH range between 5.8 and 8.5 indicated that a
His residue may be involved in substrate binding. Analysis of an align
ment of all known PDC protein sequences showed two invariant His resid
ues (His113 and His114) which, according to the crystal structure of y
east PDC, are in the vicinity of the active site. Here we demonstrate
that replacement of His114 by Gin does not have a great effect on cofa
ctor and substrate binding. However, the k(cat) is decreased indicatin
g that His114, may assist in catalysis. In contrast, substitution of H
is113 by Gin renders the enzyme completely inactive. This mutant has d
ecreased affinity for both cofactors, as revealed by measurements of t
ryptophan fluorescence quenching. However, this decreased affinity is
insufficient to account for the complete loss of activity. Despite its
inability to support overall catalysis, this [Gln113]PDC mutant is ca
pable of releasing acetaldehyde from 2-(1-hydroxyethyl)thiamin diphosp
hate supplied exogenously. It is proposed that upon substrate binding,
His113 is placed close to C2 of the thiazole ring. Subsequent deproto
nation of this atom leads to a conformational change that allows a fle
xible loop (residues 105-112) that precedes His113 to close over the a
ctive site. Hence, replacement of His113 by another residue interferes
with this closure of the active site and thus disrupts the catalytic
process.