Site-directed mutagenesis of residues at subunit interfaces of porcine fructose-1,6-bisphosphatase.
Mutation of Arg-15, Glu-19, Arg-22, and Thr-27 of porcine liver fructose-1,6-bisphosphatase was carried out by site-directed mutagenesis. These residues are conserved in all known primary sequences of mammalian fructose-1,6-bisphosphatase. On the basis of the crystal structure of the enzyme, Arg-15, Glu-19, and Arg-22 are located at the interface of the two dimers (C1-C2 and C3-C4), and Thr-27 is in the AMP binding site. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible differences were observed between the secondary structures of the wild-type and mutant forms of fructose-1, 6-bisphosphatase on the basis of CD data. Kinetic analyses revealed similar kcat values for mutants R15A, E19Q, R22K, and T27A of fructose-1,6-bisphosphatase; however, a 2-fold increase of kcat was observed with R22M compared with that of the wild-type enzyme. Small changes in Km values for fructose-1,6-bisphosphate were found in the five mutants. 4 6-fold decreases in Ki values for fructose 2,6-bisphosphate and 5-9-fold decreases in the binding affinity of Mg2+ relative to the wild-type enzyme were exhibited by R15A and E19Q. No alteration of Mg2+ cooperativity was found in the five mutants. Significant changes in Ki values for AMP were obtained in the case of R22K (30-fold) and T27A (1300-fold) with a Hill coefficient of 2.0. Replacement of Arg-22 with methionine, however, caused the total loss of AMP cooperativity without changing AMP affinity. Modeling of the mutant structures was undertaken in an attempt to define the functional role of Arg-22. These studies link specific interactions between subunits in fructose-1,6-bisphosphatase to observed properties of cooperativity.
Shyur
L F
LF
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Aleshin
A E
AE
Honzatko
R B
RB
Fromm
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D017931:DNA Primers; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D013912:Threonine
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013912:Threonine; D018698:Glutamic Acid; D001120:Arginine; D006597:Fructose-Bisphosphatase