Roles and Microenvironments of Tryptophanyl Residues of Spinach Phosphoribulokinase

• Published in: Archives of biochemistry and biophysics 1998-04, Vol.352 (1), p.130-136
• Main Authors: Brandes, Hillel K., Larimer, Frank W., Lu, Tse-Yuan S., Dey, Joykrishna, Hartman, Fred C.
• Format: Article
• Language: English
• Subjects: active site; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; AZUCARES FOSFATOS; Base Sequence; BINDING SITES; Binding Sites - genetics; DNA Primers - genetics; ENZYMIC ACTIVITY; FLUORESCENCE; FLUORESCENCIA; Kinetics; Molecular Sequence Data; MUTACION; mutagenesis; Mutagenesis, Site-Directed; MUTATION; phosphoribulokinase; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RIBULOSE 1,5-DIPHOSPHATE; Sequence Homology, Amino Acid; SITE-DIRECTED MUTAGENESIS; Spectrometry, Fluorescence; SPINACIA OLERACEA; Spinacia oleracea - enzymology; Spinacia oleracea - genetics; SUCRE PHOSPHATE; SUGAR PHOSPHATES; TARGETED MUTAGENESIS; TRANSFERASAS; TRANSFERASE; TRANSFERASES; TRIPTOFANO; TRYPTOPHAN; Tryptophan - chemistry; TRYPTOPHANE
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1998.0580

Phosphoribulokinase is one of several Calvin cycle enzymes that are light-regulated via the ferredoxin–thioredoxin system (R. A. Wolosiuk and B. B. Buchanan, 1978,Arch. Biochem. Biophys.189, 97–101). Substitution of the only two Trp residues of the enzyme was prompted by the following goals: to identify each tryptophanyl residue with respect to prior classifications as exposed and buried (C. A. Ghironet al.,1988,Arch. Biochem. Biophys.260, 267–272); to explore the possible active-site location and function of conserved Trp155, as suggested by sequence proximity to catalytic Asp160 (H. A. Charlieret al.,1994,Biochemistry33, 9343–9350); and to determine if fluorescence of a Trp residue can serve as a gauge of conformational differences between the reduced (active) and the oxidized (inactive) forms of the enzyme. Emission spectra and acrylamide quenching data demonstrate that Trp155 is solvent exposed, while Trp241 is buried. Kinetic parameters of the W241F mutant are not significantly altered relative to those of wild-type enzyme, thereby discounting any requirement for Trp at position 241. While substitution of Trp155 with Phe or Ala has little impact onVmax, theKmfor Ru5P and ATP are increased substantially; the diminished affinity for ATP is particularly pronounced in the case of the Ala substitution. In further support of an active-site location of Trp155, its fluorescence emission is subject to quenching by nucleotides. Fluorescence quenching of reduced W241F by ATP gives a dissociation constant (Kd) of 37 μM, virtually identical with itsKmof 46 μM, and provides for the first time a direct measurement of the interaction of the kinase with product ADP (Kdof 1.3 mM). Fluorescence quenching of oxidized W241F by ATP reveals aKdof 28 mM; however, this weakened binding does not reflect an altered microenvironment of Trp155, as its steady-state emission and fluorescence lifetimes are unaffected by the oxidation state.