Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each approximately 90-kDa subunit contains active-site pyridoxal 5'-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133-->Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at approximately 45 degrees C. They are manifested by slight increases in unordered structure and (1)H/(2)H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at approximately 45 degrees C without prior release into solution of pyridoxal 5'-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 degrees C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 degrees C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a approximately 15 degrees C higher value on the temperature scale. Apart from greater stability, the Asn-133-->Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.