An Active-Site Bro̷nsted Acid-Base Catalyst Destabilizes Mandelate Racemase and Related Subgroup Enzymes:...

Area of Science:

• Biochemistry
• Enzymology
• Protein Stability

Background:

• Enzymes of the enolase superfamily (ENS) share a conserved mechanism for α-proton abstraction.
• Divergent evolution has led to diverse functions within the ENS.
• The mandelate racemase (MR)-subgroup of ENS enzymes utilizes active-site Lys and His residues as Bro̷nsted acid-base catalysts.

Purpose of the Study:

• To investigate the contribution of Bro̷nsted acid-base catalysts to the thermostability of four MR-subgroup enzymes.
• To understand how mutations in catalytic residues affect enzyme stability and potentially catalytic mechanisms.

Main Methods:

• Differential scanning calorimetry (DSC) was used to measure the melting temperature (T_m) of wild-type and mutant enzymes.
• Mutagenesis was employed to substitute key residues (Lys and His) in the active sites of MR-subgroup enzymes.
• Four enzymes were studied: MR, d-tartrate dehydratase, l-talarate/galactarate dehydratase, and l-fuconate dehydratase.

Main Results:

• Mutations from Lys-Lys-X (KxK) to Lys-X-Met (KxM) increased thermostability in all four enzymes, most notably in MR (ΔT_m = +8.6 °C).
• Mutations from KxK to Met-X-Lys (MxK) decreased thermostability across all tested enzymes.
• Substitution of His with Asn significantly stabilized only MR, while having less effect on other enzymes.

Conclusions:

• The active sites of MR-subgroup enzymes are destabilized by the Lys Bro̷nsted acid-base catalyst.
• This active-site destabilization may be energetically coupled to conformational changes required for catalysis upon substrate binding.
• Understanding these stability-function relationships provides insights into enzyme evolution and catalytic mechanisms.