Isolation and characterization of a photoaffinity labeled peptide from the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes for the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. In the absence of activation light, binding studies demonstrate that the o-aryl azide competes with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymate activity was lost when enzyme was irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-(3)H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sties per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate furnished partial protection and geranyl pyrophosphate provided no protection. Isolation of modified peptides from photoaffinity labeled enzyme provides the most convincing evidence for catalytic site specific labeling. Since a single CNBr fragment contains 80% of the label, this fragment of 30 amino acid residues has been isolated and characterized from both labeled and unlabeled enzyme. Several lines of evidence indicate that a number of amino acids from this CNBr fragment have been modified. First, peptide maps of trypsin digest of this peptide or phoaffinity labeled enzyme indicate that the attached label is distributed amongst at least 3 of the resulting tryptic peptides. Second, a comparison of amino acid analysis of this CNBr fragment with its counterpart isolated from native enzyme also suggests that a number of amino acids have been modified rather than a few by the photoaffinity labeling process. Third, 2-dimensional chromatography of the pronase digest of the labeled CNBr fragment verifies that at least 11 different products resulted from photoaffinity labeling. Finally, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most frequently modified amino acids are a specific arginine and alanine.