The Moderately (D)efficient Enzyme: Catalysis-Related Damage In Vivo and Its Repair

Abstract

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo, and why.

Why cutting respiratory CO2 loss from crops is possible, practicable, and prudential

“…Expressing enzyme working life as a CCR value is critical because many enzymes are subject to selfinactivation by damage from the very reaction that they catalyse, so that the more reactions an enzyme molecule performs, the likelier it is to die. 27,40 Thus, finding enzymes that often die young, that is, have unusually low CCRs, is a logical way to locate the most improvable targets; raw protein turnover rate is a less effective screen for enzyme lifespan improvability because enzymes that die young in CCR terms do not necessarily turn over especially fast. 27,40 It is the balance of metabolic flux and enzyme replacement rate that matters (Equation 1).…”

“…27,40 Thus, finding enzymes that often die young, that is, have unusually low CCRs, is a logical way to locate the most improvable targets; raw protein turnover rate is a less effective screen for enzyme lifespan improvability because enzymes that die young in CCR terms do not necessarily turn over especially fast. 27,40 It is the balance of metabolic flux and enzyme replacement rate that matters (Equation 1).…”

Respiratory energy demands and scope for demand expansion and destruction

“…As said at the outset, ∼50% of crop respiratory energy goes to support maintenance processes ( Figure 1B ), and roughly half of that 50% fuels turnover of enzymes and other proteins ( Supplemental Table S1 ). Besides being governed by developmental stage, environmental conditions, misfolding, and general nonenzymatic damage reactions such as carbonylation and nitrosylation ( Nelson and Millar, 2015 ), enzyme turnover can be driven in a more specific way by self-inactivation resulting from chemical damage done to the enzyme by its own reaction mechanism, substrates, or products ( Bathe et al, 2021 ; Hanson et al, 2021 ; Colinas and Fitzpatrick, 2022 ). Extreme examples are suicide enzymes, which self-inactivate after mediating a single catalytic cycle, for example, the thiazole synthase THI4 ( Joshi et al, 2020 ).…”

“…A suicide enzyme like THI4 has a CCR of one and very short-lived enzymes have CCRs of tens to hundreds, but CCRs in Arabidopsis ( Arabidopsis thaliana ) range up to >10 7 and the median value is 4 × 10 5 ( Hanson et al, 2021 ). The high energy cost of protein turnover (≥6.3 ATP per residue, see above) makes abundant enzymes with low CCRs particularly large items in the maintenance energy budget, and hence prime targets for energy demand destruction by extending their working lives ( Bathe et al, 2021 ). There may well be considerable scope for such life-lengthening.…”

“… McCullough and Hunt, 1989 ; Earl and Tollenaar, 1998 ). Second, such improvement has been demonstrated; certain natural enzymes have residues favoring self-inactivation that can be mutated to other residues that extend life but hardly affect catalytic activity ( Bathe et al, 2021 ). Third, the lifespan of low-CCR enzymes prone to catalytic self-inactivation can in principle be lengthened via continuous directed evolution in yeast or Escherichia coli since damage that an enzyme does to itself will occur whether the enzyme is in its native host or a foreign host ( Hanson et al, 2021 ; García-García et al, 2022 ).…”