title: Contribution of histidine residues to the conformational stability of ribonuclease T1 and mutant Glu-58----Ala.
abstract: The pK values of the histidine residues in ribonuclease T1 (RNase T1) are unusually high: 7.8 (His-92), 7.9 (His-40), and 7.3 (His-27) [Inagaki et al. (1981) J. Biochem. 89, 1185-1195]. In the RNase T1 mutant Glu-58----Ala, the first two pK values are reduced to 7.4 (His-92) and 7.1 (His-40). These lower pKs were expected since His-92 (5.5 A) and His-40 (3.7 A) are in close proximity to Glu-58 at the active site. The conformational stability of RNase T1 increases by over 4 kcal/mol between pH 9 and 5, and this can be entirely accounted for by the greater affinity for protons by the His residues in the folded protein (average pK = 7.6) than in the unfolded protein (pk approximately 6.6). Thus, almost half of the net conformational stability of RNase T1 results from a difference between the pK values of the histidine residues in the folded and unfolded conformations. In the Glu-58----Ala mutant, the increase in stability between pH 9 and 5 is halved (approximately 2 kcal/mol), as expected on the basis of the lower pK values for the His residues in the folded protein (average pK = 7.1). As a consequence, RNase T1 is more stable than the mutant below pH 7.5, and less stable above pH 7.5. These results emphasize the importance of measuring the conformational stability as a function of pH when comparing proteins differing in structure.
authors.affiliation: Biochemistry Department, Texas A&M University, College Station 77843.
authors.forename: M
authors.initials: M
authors.lastname: McNutt
authors.forename: L S
authors.initials: LS
authors.lastname: Mullins
authors.forename: F M
authors.initials: FM
authors.lastname: Raushel
authors.forename: C N
authors.initials: CN
authors.lastname: Pace
mesh_terms: D000409:Alanine / Q000235:genetics*; D004795:Enzyme Stability; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D006163:Ribonuclease T1; D000409:Alanine