Stability, activity and structure of adenylate kinase mutants.
Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.
Spuergin
P
P
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Abele
U
U
Schulz
G E
GE
D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003196:Computer Graphics; D018360:Crystallography, X-Ray; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D007093:Imidazoles / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D015226:Dinucleoside Phosphates; D006146:Guanidines; D007093:Imidazoles; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C029899:imidazole; D000263:Adenylate Kinase; D019791:Guanidine