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 (lambdaL), at position 48, was biosynthetically replaced by the analogue 1,2,4-triazole-3-alanine (Taz), the basicity of which is 3 pKa 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-lambdaL and Taz48-lambdaL 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-lambdaL is less stable than His48-lambdaL by approximately 3.5 kcal/mol, probably as a result of the loss of a hydrogen bond in the native form of Taz48-lambdaL. Lowering the pH leads to a progressive stabilization of Taz48-lambdaL relative to His48-lambdaL because of the abnormally low pKa of His48 in the native form of His48-lambdaL.
Soumillion
P
P
Université Catholique de Louvain, Laboratoire de Biochimie Physique et des Biopolymères, Louvain-la-Neuve, Belgium.
Fastrez
J
J
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; C003633:triazolealanine; D006639:Histidine; D009113:Muramidase; D000409:Alanine