Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.
Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of these reagents. Carboxypeptidase-A treatment of rabbit-muscle and T. brucei aldolase inhibited the activity of both enzymes towards fructose-1,6-bisphosphate (Fru(1,6)P2), while the activity towards fructose-1-phosphate (Fru-1-P) was affected only in the case of the trypanosomal enzyme. Moreover carboxypeptidase-A treatment reduced the turnover numbers of these two aldolases for both Fru(1,6)P2 and Fru-1-P to a similar level. Glyceraldehyde 3-phosphate, in the absence of dihydroxyacetone phosphate, also inactivated aldolases from rabbit muscle and T. brucei with second order rate constants of 1054 and 254 min-1 M-1, respectively. Using 5,5'-dithiobis-(2-nitrobenzoic acid) with rabbit-muscle aldolase, a total of 4 thiol groups could be titrated per subunit, resulting in a total inactivation. The presence of substrate completely protected the enzyme from inactivation. Methyl methanethiosulfonate also reacted with four cysteine residues, but this led to very little inactivation. This indicates that the inactivation by modification with DTNB is due to conformational changes in the enzyme. In T. brucei aldolase only one thiol group could be titrated with methyl methanesulfonate and there was no loss of activity. With 5,5'-dithiobis-(2-nitrobenzoic acid) five cysteines were titrated with an immediate and complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Callens
M
M
Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Opperdoes
F R
FR
D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002268:Carboxypeptidases / Q000494:pharmacology; D043422:Carboxypeptidases A; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005986:Glyceraldehyde 3-Phosphate / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D011817:Rabbits; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology*; D013438:Sulfhydryl Compounds; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology*
D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D005986:Glyceraldehyde 3-Phosphate; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D005634:Fructose-Bisphosphate Aldolase; D003545:Cysteine