Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protei n in which each approximate to 90-kDa subunit contains active-site pyridoxa l 5'-phosphate. To unravel factors contributing to the stability of MalP, t hermal denaturations of wild-type MalP and a thermostable active-site mutan t (Asn-133 --> Ala) were compared by monitoring enzyme activity, cofactor d issociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at approximate to 45 degrees C. They are manifested by slight i ncreases in unordered structure and H-1/H-2 exchange, and reflect reversibl e inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at approximate to 45 degrees C without p rior release into solution of pyridoxal 5'-phosphate. It is driven by elect rostatic rather than hydrophobic interactions between MalP dimers, and lead s to irreversible inactivation of the enzyme. Aggregation is inhibited effi ciently and specifically by oxyanions such as phosphate, and AMP which ther efore, stabilize MalP against the irreversible denaturation step at 45 degr ees C. Melting of the secondary structure in soluble and aggregated MalP ta kes place at much higher temperatures of approx. 58 and 67 degrees C, respe ctively. Replacement of Asn-133 by Ala does not change the mechanism of the rmal denaturation, but leads to a shift of the entire pathway to a approxim ate to 15 degrees C higher value on the temperature scale. Apart from great er stability, the Asn-133-->Ala mutant shows a 2-fold smaller turnover numb er and a 4.6-fold smaller energy of activation than wild-type MalP, probabl y indicating that the site-specific replacement of Asn-133 brings about a g reater rigidity of the active-site environment of the enzyme. A structure-b ased model is proposed which explains the stabilizing interaction between M alP and oxyanions, or AMP.