Significance of the amino terminus of rat testis fructose-6-phosphate, 2-kinase:fructose-2,6-bisphosphatase.
In order to assess the functional properties of the extreme amino-terminal peptide of rat testis fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase, 24 and 30 amino acids from the NH2 terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type (WT) and the mutant (Del 24 and Del 30) enzymes were purified to homogeneity. These enzymes were homodimers of subunit M(r) values 54,000, 51,300, and 50,600. Deletion of 1-24 and 1-30 residues resulted in over 70% loss of Fru-6-P,2-kinase activity and 2-fold increase in Fru-2,6-bisphosphatase. KmFru-6-P increased 7.5-fold, but there was no significant change in KmFru-2,6P2 of the phosphatase. These mutant enzymes were more susceptible to protein concentration-dependent dissociation and thermal inactivation. In 2 M urea the kinase and the phosphatase activities of WT increased 17 and 56%, respectively, but no activation of any of the mutant enzymes was observed. At higher urea concentrations, all the enzymes were inactivated. Unfolding in urea, followed with intrinsic tryptophan fluorescence, showed that the unfolding proceeded in at least two stages. Fluorescence intensity at 338 nm of all these enzymes decreased below 2 M urea in which the enzyme activities remained the same (Del 24) or activated (WT). The second stage of unfolding was seen as the maximum shifted to 342 nm in 2-4 M urea, presumably involving dissociation. Complete unfolding above 4 M urea resulted in further shift in fluorescence maximum to 350 nm.
Research Service, Department of Veterans Affairs Medical Center, Dallas, Texas.
N
N
Tominaga
Y
Y
Minami
R
R
Sakakibara
K
K
Uyeda
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D013050:Spectrometry, Fluorescence; D013737:Testis / Q000201:enzymology*; D014508:Urea / Q000737:chemistry
D004277:DNA, Single-Stranded; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D014508:Urea; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases