@article { ISI:000458407700015,
	title = {The pK(a) values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme},
	journal = {PROTEIN SCIENCE},
	volume = {28},
	number = {3},
	year = {2019},
	month = {MAR},
	pages = {620-632},
	abstract = {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 (Kuroki et al., PNAS 1999; 96:8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pK(a) values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pK(a) value of the putative nucleophile His26 is 6.8 +/- 0.1, whereas that of the general acid Glu11 is 4.7 +/- 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions. Brief statement The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pK(a) values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild-type and mutant form of T4 lysozyme cleave bacterial peptidoglycan.},
	issn = {0961-8368},
	doi = {10.1002/pro.3562},
	author = {Brockerman, Jacob A. and Okon, Mark and Withers, Stephen G. and McIntosh, Lawrence P.}
}
