IDENTIFICATION OF CATALYTIC BASES IN THE ACTIVE-SITE OF ESCHERICHIA-COLI METHYLGLYOXAL SYNTHASE - CLONING, EXPRESSION, AND FUNCTIONAL-CHARACTERIZATION OF CONSERVED ASPARTIC-ACID RESIDUES
D. Saadat et Dht. Harrison, IDENTIFICATION OF CATALYTIC BASES IN THE ACTIVE-SITE OF ESCHERICHIA-COLI METHYLGLYOXAL SYNTHASE - CLONING, EXPRESSION, AND FUNCTIONAL-CHARACTERIZATION OF CONSERVED ASPARTIC-ACID RESIDUES, Biochemistry, 37(28), 1998, pp. 10074-10086
Methylglyoxal synthase provides bacteria with an alternative to triose
phosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP)
. In the present studies, the methylglyoxal synthase gene in Escherich
ia coli has been cloned and sequenced. The identified open reading fra
me (ORF) codes for a polypeptide of 152 amino acids, consistent with t
he 17 kDa purified protein. The sequence of this protein is not simila
r to any other protein of known function, including the functionally s
imilar protein triosephosphate isomerase. The methylglyoxal synthase g
ene was amplified by PCR, subcloned into the pET16B expression vector,
and expressed in the host E, coli BL21(DE3). Sequence comparison of t
he methylglyoxal protein and related ORFs from four different bacteria
l species revealed that four aspartic acid and no glutamic acid residu
es are absolutely conserved. The function of the four aspartic acid re
sidues was tested by mutating them to either asparagine or glutamic ac
id. Thermal denaturation, CD spectroscopy, and gel filtration experime
nts showed that the mutant enzymes had the same secondary and quaterna
ry structure as the wild-type enzyme. Kinetic characterization of both
Asp 71 and Asp 101 mutant proteins shows reduced k(cat)/K-m by 10(3)-
and 10(4)-fold respectively, suggesting that they are both intimately
involved in catalysis, A time-dependent inhibition of both Asp 20 and
Asp 91 asparagine mutants by DHAP suggests that these two residues ar
e involved with protecting the enzyme from DHAP or reactive intermedia
tes along the catalytic pathway. In combination with the results of a-
phosphoglycolate binding studies, a catalytic mechanism is proposed.