4e97 | pdb_00004e97
T4 Lysozyme L99A/M102H with 2-Mercaptoethanol Bound
Structural highlights
Function[ENLYS_BPT4] Endolysin with lysozyme activity that degrades host peptidoglycans and participates with the holin and spanin proteins in the sequential events which lead to the programmed host cell lysis releasing the mature viral particles. Once the holin has permeabilized the host cell membrane, the endolysin can reach the periplasm and break down the peptidoglycan layer.[1] Publication Abstract from PubMedSynthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 --> His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M) = 0.43 M(-1) s(-1) and a (k(cat)/K(M))/k(uncat) = 10(7) at pH 5.0. The crystal structure of this enzyme was determined at 1.30 A, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments. Engineering a model protein cavity to catalyze the Kemp elimination.,Merski M, Shoichet BK Proc Natl Acad Sci U S A. 2012 Sep 17. PMID:22988064[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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