Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase.
The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.
Roeske
C A
CA
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Kutny
R M
RM
Budde
R J
RJ
Chollet
R
R
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010769:Phosphothreonine / Q000032:analysis*; D010770:Phosphotransferases / Q000302:isolation & purification*; D010940:Plant Proteins / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D011772:Pyruvate, Orthophosphate Dikinase / Q000302:isolation & purification*; D013912:Threonine / Q000031:analogs & derivatives*; D003313:Zea mays / Q000201:enzymology
D010940:Plant Proteins; D010769:Phosphothreonine; D013912:Threonine; D010770:Phosphotransferases; C037396:PPDK protein, Zea mays; D011772:Pyruvate, Orthophosphate Dikinase