The Number of Catalytic Cycles in an Enzyme’s Lifetime and Why It Matters for Bioproduction

Metabolic engineering uses enzymes as parts to build bioproduction systems for specified tasks. Although a part’s working life and failure mechanisms are key performance data in engineering, this is not yet so in metabolic engineering because it is unclear how long enzymes last in vivo or what causes them to fail or be replaced. Consequently, the energy cost of replacing enzyme parts cannot be quantified and enzymes cannot be engineered to lengthen their lives and hence lower their replacement costs. These costs are very high; the turnover of enzymes and other proteins appropriates up to half the maintenance energy budget in microorganisms and plants. Enzyme protein turnover rates therefore impact the efficiency of all bioproduction systems.

Guided by catalyst engineering, we adopted Catalytic-Cycles-till-Replacement (CCR) as a metric for an enzyme’s functional lifespan in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before it fails or is replaced, i.e. metabolic flux rate / protein turnover rate. We used estimated metabolic fluxes and measured protein turnover rates to calculate CCRs for ~100-200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis. The CCRs in these organisms had similar ranges (<10² to >10⁷) but different median values (3-4 × 10⁴ in L. lactis and yeast vs. 4 × 10⁵ in Arabidopsis). Enzymes whose substrates, products, or mechanisms can attack vulnerable active-site residues had significantly lower median CCR values in all organisms. This finding, along with published cases of mechanism-based inactivation, indicates that an important but underrecognized cause of enzyme failure is active-site damage inflicted by reaction chemistry, with vulnerability to such damage determined by which residues form the active site. It may therefore be feasible to engineer enzymes to raise CCR and lower replacement costs to enhance overall bioproductivity in systems ranging from fermentations to fields and forests.