J. Biol. Chem., Vol. 263, Issue 14, 6683-6687, 05, 1988
Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase
CA Roeske, RM Kutny, RJ Budde and R Chollet
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
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.
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