Journal of Protein Chemistry

, Volume 2, Issue 5, pp 371–381 | Cite as

Fluorescence investigations of coenzyme and substrate interactions in mannitol-1-phosphate dehydrogenase ofEscherichia coli

  • Richard A. Cerione
  • Theodore ChaseJr.


Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD+ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD+ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD+, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD+-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD+; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.

Key words

dehydrogenase mannitol-1-phosphate fluorescence fructose-6-phosphate mannitol-1-phosphate pyridoxal-5-phosphate nicotinamide adenine dinucleotide 


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Copyright information

© Plenum Publishing Corporation 1983

Authors and Affiliations

  • Richard A. Cerione
    • 1
  • Theodore ChaseJr.
    • 1
  1. 1.Department of Biochemistry and Microbiology, Cook CollegeRutgers-The State University, New Jersey Agricultural Experiment StationNew Brunswick

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