The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

Abstract

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis. CCRs in these organisms had similar ranges (<103 to >107) but different median values (3–4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

In vitro turnover numbers do not reflect in vivo activities of yeast enzymes

“…Moreover, by correlating k app of each condition with in vitro k cat we found most correlations to be poor in log scale ( Dataset S8 ). A recent study, using another proteomic dataset and a different GEM, also showed a poor correlation in log scale of R 2 = 0.27 between yeast k app and in vitro k cat at one condition ( 12 ).…”

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

“…Turnover of another rapidly degrading protein, thiamin synthase (THI1), is explained by its suicide mechanism that means the enzyme has a single catalytic cycle before it is inactivated and needs to be replaced (Chatterjee et al, 2011;Joshi et al, 2020). Recently we showed across a wide range of enzymes in Arabidopsis, yeast and bacteria, that the number of catalytic cycles until replacement varied according to the chemical risk of the reaction they undertook, including enzymes with photoactivatable substrates or with reactive oxygen producing roles in metabolism (Hanson et al, 2021).…”

“…The increased turnover of redox shuttling systems, namely glutathione and thioredoxin linked systems and the MDH enzymes involved in malate shuttles throughout the cell (Fig 4 ), may be due to increased flux through these pathways and thus a consequence of an increased rate of wear-out damage of these enzymes (Hanson et al, 2021;Tivendale et al, 2021).…”

Functional and protective hole hopping in metalloenzymes

“…TTN values range from 1 (for suicide “enzymes” 131 ) to >10 7 . 132 No accepted scale of risk factors for enzyme reactivity has been established, although an early study suggested that reactions with O 2 or H 2 O 2 increased the risk of enzyme deactivation. 133 A more recent attempt to classify enzyme reaction risks suggested that radical mechanisms and those involving highly reactive intermediates increased the likelihood of enzyme deactivation.…”