Incorporation of 1,2,4-triazole-3-alanine into a mutant of phage lambda lysozyme containing a single histidine

The only histidine residue in the H31N-H137N double mutant of phage lambda lysozyme (lambda L), at position 48, was biosynthetically replaced by the analogue 1,2,4-triazole-3-alanine (Taz), the basicity of which is 3 pK(a) units lower. A histidine-auxotrophic strain was grown to stationary phase by histidine limitation in a synthetic medium, then Taz was added on induction to produce a lysozyme with approximately 75% incorporation. The Taz-containing enzyme precipitated selectively from the cytoplasm and was purified after renaturation. Replacement by Taz had only very minor effects on the activity-pH profile of the enzyme, in contrast with the great perturbations observed for the Asn48 mutant. The relative stabilities of the His48-lambda L and Taz48-lambda L mutants were also studied as a function of pH; the results are discussed with regard to the poor accessibility of His48, the low basicity of Taz and the hydrogen bonding patterns suggested by the crystal structure. At neutral pH, Taz48-lambda L is less stable than His48-lambda L by approximately 3.5 kcal/mol, probably as a result of the loss of a hydrogen bond in the native form of Taz48-lambda L. Lowering the pH leads to a progressive stabilization of Taz48-lambda L relative to His48-lambda L because of the abnormally low pK(a) of His48 in the native form of His48-lambda L.