A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation.
Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.
Morris
A J
AJ
Biology Department, Boston University, MA 02215, USA.
Davenport
R C
RC
Tolan
D R
DR
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001894:Borohydrides / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015202:Protein Engineering; D011817:Rabbits; D012545:Schiff Bases / Q000737:chemistry*
D001894:Borohydrides; D005635:Fructosediphosphates; D012545:Schiff Bases; C025364:sodium borohydride; D001120:Arginine; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine; C029063:fructose-1,6-diphosphate