Investigating the Half‐site Reactivity of a Member of the PNDOR Family of Proteins

Abstract

Half‐site reactivity is an enzyme characteristic in which the active sites of a multimeric enzyme “fire” alternately, which means that one‐half of the identical subunits is active at any one time (1). Many metabolically important enzymes, such as alcohol dehydrogenase (ADH) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), are known to exhibit half‐site reactivity (2). Two models, induced‐fit (or alternating sites cooperativity model) and preexistent asymmetry, were proposed in early literature to explain this half‐site reactivity (3). The pyridine nucleotide disulfide oxidoreductases (PNDORs) are members of a flavoprotein family that use NADH/NADPH as their reductive substrate, with most members of the PNDOR family being found in the form of homodimers. It has previously been observed that members of the PNDOR family show half‐site reactivity (via an alternating sites cooperativity model), as evidenced by subunit asymmetry (4,5): when their bound flavin coenzyme is reduced by NAD(P)H, only one subunit reduces, with the other subunit remaining oxidized. Recent data from our laboratory suggests that the CoADR from Pyrococcus horikoshii, a member of the PNDOR family, also shows half‐site reactivity.Although many enzymes with half‐site reactivity have been characterized kinetically and structurally, the cause of half‐site reactivity and its effect on enzymatic catalytic efficiency and mechanism remains unclear. We have designed asymmetrical dimers of a member of the PNDOR family which disrupts the ability of the subunits to react in an alternating fashion. Preliminary kinetic data suggests that in an asymmetrical dimer, in which one subunit has been inactivated by mutation of its redox‐active cysteine (with the other subunit remaining active), the phCoADR actually exhibits a 3‐fold increase in catalytic efficiency when compared to the wild‐type enzyme for a range of different substrates. In addition, a higher background oxidase activity is observed in the asymmetrical dimer. Our results suggest that subunit communication, in the induced fit model of half‐site reactivity, appears to slow the overall rate of the enzyme, perhaps acting as a control on enzyme activity. The mechanisms of this control will be discussed.Support or Funding InformationThis project is supported by NSF award MCB 1518306.