Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli.
Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.
Teschner
W
W
Laboratoire d'Enzymologie Centre, National de la Recherche Scientifique, Gif-Sur-Yvette, France.
Serre
M C
MC
Garel
J R
JR
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D005632:Fructose; D008353:Mannitol; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D005947:Glucose