Lysine-146 of rabbit muscle aldolase is essential for cleavage and condensation of the C3-C4 bond of fructose 1,6-bis(phosphate).
Lysine-146 of rabbit muscle aldolase (D-fructose-1,6-biphosphate aldolase, EC 4.1.2.13) is absolutely conserved in class I (Schiff base) aldolases and has been implicated previously in catalysis by protein modification. Site-directed mutagenesis was used to change lysine-146 to alanine, glutamine, leucine, or histidine, creating the mutant enzymes K146A, K146Q, K146L, and K146H, respectively. These mutant proteins were expressed at high levels in bacteria and were purified by substrate affinity elution from CM-Sepharose, the same method that is used for the wild-type enzyme. The mutants K146A, K146Q, and K146L had substrate cleavage rates below standard detection levels. Modified cleavage assays indicated that these enzymes were (0.5-2) x 10(6)-fold decreased in the rate of catalysis of fructose 1,6-bis(phosphate) (Fru-1,6-Pa)cleavage. The K146H enzyme, however, was approximately 2000-fold slower than wild type in the rates of both cleavage and condensation of Fru-1,6-P2. In assays for the presence of enzymatic intermediates, all of the mutant enzymes were able to catalyze formation of the carbanion intermediate with dihydroxyacetone phosphate, whereas this intermediate was below the level of detection with Fru-1,6-P2. Single-turnover experiments with these enzymes in excess over radiolabeled Fru-1,6-P2 were used to measure the rates of Schiff base and product formation. The rate of Schiff base formation was decreased in each of the mutant enzymes, yet the magnitude of this decrease was less than the reduction in the respective kcat. These mutations had a much larger effect, however, on the rate of C3-C4 bond breaking, showing that Lys-146 is crucial at this step of the catalytic cycle.
Morris
A J
AJ
Biology Department, Boston University, Massachusetts 02215.
Tolan
D R
DR
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003062:Codon / Q000737:chemistry / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011817:Rabbits; D012545:Schiff Bases / Q000737:chemistry
D003062:Codon; D005635:Fructosediphosphates; D009838:Oligodeoxyribonucleotides; D012545:Schiff Bases; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine; C029063:fructose-1,6-diphosphate