Phosphoribulokinase: isolation and sequence determination  of the cysteine-containing active-site peptide modified  by 5'-p-fluorosulfonylbenzoyladenosine.
Phosphoribulokinase (ATP: D-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) is stoichiometrically inactivated by the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The inactivation is reversed upon incubation with dithiothreitol suggesting that a cysteine is modified. Since the adduct formed upon enzyme inactivation is unstable to normal procedures for peptide analysis, the site of modification has been identified by converting the labile adduct to the well-characterized carboxymethylcysteine. The approach takes advantage of the sensitivity of the thiolsulfonyl-containing adduct to reducing agents; thus the adduct acts as a temporary protecting group while the previously unmodified cysteines are blocked. The FSBA-modified cysteine is then unmasked with dithiothreitol and radiolabeled. DEAE and reverse-phase high-pressure liquid chromatography of tryptic digests indicate that a single peptide is radiolabeled. Amino-acid analysis and automated Edman degradation techniques have been employed to confirm cysteine as the site of modification. The sequence of the tryptic peptide was determined to be: Ser.Gln-GIn-Gln-Thr-Ile-Val-Ile-Gly-Leu-Ala-Ala.Asp-Ser-Gly-CM-Cys-Gly-Lys. This sequence is identical with the reported N-terminal sequence, thus identifying Cys-16 as the site of modification.
Krieger
T J
TJ
Mende-Mueller
L
L
Miziorko
H M
HM
D000241:Adenosine / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein
D000345:Affinity Labels; D009842:Oligopeptides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D000241:Adenosine; D003545:Cysteine