“Characterizing a Peculiar Mutant of T4 Lysozyme”, by Jacob Brockerman, PhD Candidate, McIntosh Lab.
T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate. To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or ionization states of all Asp, Glu, His, and Arg residues in the T4L mutant. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. These studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions.
Monday, April 1, 2019 @ 3:00pm, LSC #3, 2350 Health Sciences Mall