Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity.
Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.
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Laboratory of Molecular Neurobiology, W.M. Burke Medical Research Institute, Cornell University Medical College, White Plains, NY 10605.
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D000818:Animals; D001665:Binding Sites; D002421:Caudate Nucleus / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008961:Models, Structural; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D010450:Endopeptidases; D014357:Trypsin