Award Date

5-2014

Degree Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Chemistry

Advisor 1

Gary Kleiger

Advisor 2

Andrew Hanson

Advisor 3

Ernesto Abel-Santos

Number of Pages

23

Abstract

Ubiquitin-conjugating enzymes (E2s) covalently modify protein substrates with ubiquitins. The active site cysteine residues on E2s are essential for catalyzing the transfer of ubiquitin from the E2 active site onto the protein substrate, however there is a limited amount of information available concerning additional active site residues for E2s that may also participate in catalysis. Cdc34 is an essential E2 that has merited the lion’s share of attention for biochemical analysis of the E2 family. Previous phylogenetic analysis of Cdc34 amino acid sequences has identified an invariably conserved histidine residue close to the active site cysteine in the primary structure, however whether this residue actually participates in Cdc34 function is unknown. Here we demonstrate that histidine 98 on the human Cdc34 ubiquitin conjugating enzyme is vital to the enzymatic activity of the E2. Recombinant His98Ala mutant Cdc34 was isolated from bacterial cells engineered to express the protein and compared to wild-type Cdc34 through two complementary ubiquitination assays. Substitution of the histidine residue with alanine resulted in a nearly complete loss of function. These results uncover the possible roles that histidine 98 may play during Cdc34 function.

Keywords

Amino acid sequence; Bioconjugates; Enzymes; Enzymes — Regulation; Ubiquitin

Disciplines

Biochemistry | Chemistry

Language

English

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