JBC, Vol. 250, Issue 20, 8062-8068, Oct, 1975
Evidence for essential lysyl residues in ribulosebisphosphate carboxylase by use of the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate
I. L. Norton, M. H. Welch and F. C. Hartman
A previous study from our laboratory suggested that
3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is an affinity label for
spinach ribulosebisphosphate carboxylase. To identify the essential
residues that react with the reagent we have isolated and characterized the
labeled peptides that are present in tryptic digests of inactivated enzyme
but lacking in digests of the substrate-protected enzyme. Peptides
representing two sites of modification have been obtained from the
inactivated carboxylase. Both sites of reaction have been identified as
lysyl residues based on the conversion of the derivatives to free lysine by
oxidation with sodium metaperiodate. Sodium dodecyl sulfate-gel
electrophoretic experiments show that both essential lysyl residues are
contained within the large subunit of ribulosebisphosphate carboxylase. In
addition to lysyl residues, sulfhydryl groups of the carboxylase are also
modified, but their modification seems to play little role in the
inactivation process. The carboxylase modified in the presence of substrate
contains sulfhydryl derivatives but is essentially lacking in lysyl
derivatives. By comparing the profiles from ion exchange chromatography of
labeled peptides in digests of inactivated and substrate-protected enzyme,
we conclude that the same sulfhydryl groups are modified in the absence and
presence of substrate.
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