Site-directed mutagenesis identifies aspartate 33 as a previously unidentified critical residue in the catalytic mechanism of rabbit aldolase A.
The expression and purification of the rabbit muscle aldolase A (D-fructose 1,6-bisphosphate:D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from an expression plasmid in bacteria is described. The enzyme is produced in bacteria at a level of 300 mg/liter and is indistinguishable from the enzyme isolated from muscle in assays using fructose 1,6-bisphosphate and fructose 1-phosphate. The recombinant enzyme has the same primary, secondary, and quaternary structure as the muscle enzyme. Aspartic acid 33, found near the active site lysine in the crystal structure, is changed to alanine, serine, and glutamic acid by site-directed mutagenesis, resulting in the mutant proteins, D33A, D33S, and D33E, respectively. The mutant enzymes are purified by substrate affinity elution from carboxylmethyl-Sepharose, the same method as that used for the wild-type enzyme. The secondary and quaternary structure of D33A is identical to wild-type aldolase when analyzed by light scattering, gel filtration, and circular dichroism. Moreover, the hexose substrate can be fixed in the active site by reduction of the Schiff base with sodium borohydride, indicating that the active site is not drastically altered. These single mutations in the active site have a serious effect on the activity of the enzyme. In addition, the rate of carbanion oxidation for D33A is 17-29 times slower when the substrate is fructose 1,6-bisphosphate versus dihydroxyacetone phosphate, whereas in the wild-type there is no significant difference in these rates. This evidence and the conservation of this residue in other class I aldolases indicate that aspartic acid 33 is an essential residue in the catalytic mechanism, possibly involved in abstraction of the carbon 4 hydroxyl proton.
Morris
A J
AJ
Biology Department, Boston University, Massachusetts 02215.
Tolan
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D005634:Fructose-Bisphosphate Aldolase