Enzyme Stability-Activity Trade-Off: New Insights from Protein Stability Weaknesses and Evolutionary Conservation

Abstract

A general limitation of the use of enzymes in biotechnological processes under sometimes nonphysiological conditions is the complex interplay between two key quantities, enzyme activity and stability, where the increase of one is often associated with the decrease of the other. A precise stability-activity trade-off is necessary for the enzymes to be fully functional, but its weight in different protein regions and its dependence on environmental conditions is not yet elucidated. To advance this issue, we used the formalism that we have recently developed to effectively identify stability strength and weakness regions in protein structures and applied it to a large set of globular enzymes with known experimental structure and catalytic sites. Our analysis showed a striking oscillatory pattern of free energy compensation centered on the catalytic region. Indeed, catalytic residues are usually nonoptimal with respect to stability, but residues in the first shell around the catalytic site are, on the average, stability strengths and thus compensate for this lack of stability; residues in the second shell are weaker again, and so on. This trend is consistent across all enzyme families. It is accompanied by a similar, but less pronounced, pattern of residue conservation across evolution. In addition, we analyzed cold- and heat-adapted enzymes separately and highlighted different patterns of stability strengths and weaknesses, which provide insight into the longstanding problem of catalytic rate enhancement in cold environments. The successful comparison of our stability and conservation results with experimental fitness data, obtained by deep mutagenesis scanning, led us to propose criteria for improving catalytic activity while maintaining enzyme stability, a key goal in enzyme design.

FiTMuSiC: leveraging structural and (co)evolutionary data for protein fitness prediction

“…This is not surprising given deleteriousness and fitness are very well correlated, while stability and fitness are less so. For example, all functional residues are highly important for fitness while very poorly optimized for stability [ 40 ].…”

“…It is well known that enzymes exhibit an activity-stability trade-off: residues in catalytic regions are optimized for functional reasons and less or not at all for stability, while other residues are very important for protein folding and stability and play little to no role in function [ 40 ]. FiTMuSiC can help in distinguishing these functional and structural contributions.…”

“…Although stability and fitness are imperfectly correlated [ 40 ], we also submitted a prediction model based on a rescaling of the score of the thermodynamic stability predictor PoPMuSiC (POP): where the parameters and were identified on the same training set as the other models. The functions bound the output between 0 and 1.…”

Rheostatic contributions to protein stability can obscure a position's functional role

“…Because we observed variation in both specific activity (Figure 3c) and T m‐app (Figure 6b), we next explored the relationship between the two parameters. In the enzyme engineering field, a “stability‐activity trade‐off” is generally expected, i.e., substitutions that improve enzyme specific activity are often expected to decrease enzyme stability (Hou et al, 2023; Khersonsky et al, 2010; Klesmith et al, 2017; Xie et al, 2022; Xie & Warshel, 2022). Thus, we assessed the correlation between thermal stability and specific activity for ZmPYK substitution sets at positions H48X, V169X, and F446X.…”

Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function

“…Interestingly, for enzymes, there is a widespread expectation of a “stability/activity tradeoff” ( e.g. , ( 131 , 132 , 133 , 134 )). However, Whitehead et al.…”