The role of quaternary interactions on the stability and activity of ascorbate peroxidase

• Published in: Protein science Vol. 7; no. 10; pp. 2089 - 2098
• Main Authors: Mandelman, David, Li, Huiying, Poulos, Thomas L., Schwarz, Frederick P.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.10.1998
• Subjects: ascorbate peroxidase; Ascorbate Peroxidases; Ascorbic Acid - metabolism; Calorimetry, Differential Scanning; Crystallography, X-Ray; differential scanning calorimetry; Dimerization; Enzyme Stability; Hydrogen Bonding; Kinetics; Models, Molecular; Molecular Weight; Peroxidases - chemistry; Plant Proteins - chemistry; Point Mutation - genetics; Protein Conformation; Protein Denaturation; Protein Folding; quaternary structure; salt bridges; solvent structure; subunit interface; Thermodynamics; thermostability; Water - chemistry
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560071005

Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu 112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (ΔHc) with Tm = 58.3 ± 0.5 °C, 56.0 ± 0.8 °C, and 53.0 ± 0.9 °C and ΔHc = 245 ± 29 kcal/mol, 199 ± 38 kcal/mol, and 170 ± 25 kcal/mol for wild‐type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the El 12K mutant which demonstrates that the non‐Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild‐type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.