Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.
Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.
Brandes
H K
HK
Protein Engineering and Molecular Mutagenesis Program, Oak Ridge National Laboratory, Tennessee.
Stringer
C D
CD
Hartman
F C
FC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D012274:Ribulosephosphates / Q000494:pharmacology; D014357:Trypsin
D003432:Cross-Linking Reagents; D012274:Ribulosephosphates; C013936:difluorodinitrobenzene sulfone; C031524:ribulose 5-phosphate; D000255:Adenosine Triphosphate; D004139:Dinitrofluorobenzene; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D014357:Trypsin; D003545:Cysteine