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

Update Item Information
Title Isolation and characterization of a photoaffinity labeled peptide from the catalytic site of prenyltransferase.
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Brems, David Nettleship
Date 1980-08
Description 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.
Type Text
Publisher University of Utah
Subject Isolation; Purification; Lipids; Synthesis
Subject MESH Dimethylallyltranstransferase; Peptides
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Isolation and characterization of a photoaffinity labeled peptide from the catalytic site of prenyltransferase." Spencer S. Eccles Health Sciences Library. Print version of "Isolation and characterization of a photoaffinity labeled peptide from the catalytic site of prenyltransferase." available at J. Willard Marriott Library Special Collection. QD 3.5 1980 B74.
Rights Management © David Nettleship Brems.
Format application/pdf
Format Medium application/pdf
Identifier us-etd2,23719
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
ARK ark:/87278/s6qc0hzk
Setname ir_etd
ID 192118
Reference URL https://collections.lib.utah.edu/ark:/87278/s6qc0hzk
Back to Search Results