Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics
Go big or you’ll get lost
Rational mutagenesis is a common approach to investigating or engineering enzyme function in vitro, but the ease with which one can manipulate protein sequences belies many pitfalls in connecting sparse activity data to an enzyme’s true functional landscape. Using a high-throughput platform, Markin et al. expressed, purified, and performed an array of kinetic measurements on a target esterase, collecting data from >1000 mutations spanning the entire protein (see the Perspective by Baumer and Whitehead). Protein misfolding into an inactive state, rather than decreased equilibrium stability, was a crucial factor in negatively affected variants spread throughout the protein. When combined with prior mechanistic understanding and structures, four “functional components” help to rationalize the otherwise complex spatial pattern of effects of mutations on different aspects of enzyme function, all of which would be invisible from mutagenesis of just a few residues.
Structured Abstract
INTRODUCTION
Enzymes possess extraordinary catalytic proficiency and specificity. These properties ultimately derive from interactions not just between the active-site residues and the substrate but from functional interactions throughout a folded enzyme. Therefore, understanding the origins of catalytic proficiency and specificity will require the ability to make mutations throughout the protein. Traditionally, enzyme active sites have been characterized by means of site-directed mutagenesis (SDM), revealing much about the catalytic functions of these residues; nevertheless, SDM is low-throughput, costly, and labor intensive. By contrast, recently developed high-throughput mutational scanning techniques assay large numbers of sequences but provide only coarse estimates of function, such as the amount of product generated at a particular time under a particular set of conditions or overall organismal fitness.
RATIONALE
New technologies are needed to overcome the limitations of current approaches and allow deep characterization of many enzyme variants in a cost- and time-efficient manner. To meet this challenge, we developed a high-throughput microfluidic platform that allows the simultaneous expression and purification of more than 1500 rationally chosen enzyme mutants in hours and allows their quantitative functional characterization in days. HT-MEK (High-Throughput Microfluidic Enzyme Kinetics) can be used with any enzyme system that can be tagged and expressed in vitro and has a direct or coupled fluorogenic assay.
RESULTS
As a first application of HT-MEK, we functionally characterized 1036 single-site mutants that contain either a glycine or valine substitution at each position within PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), a well-studied enzyme from the alkaline phosphatase superfamily. For each mutant, we measured Michaelis-Menten kinetics [apparent unimolecular rate constant (kcat), Michaelis constant (Km), and kcat/Km] for multiple substrates, inhibition constants, and effects on folding, obtaining more than 5000 kinetic and thermodynamic constants from more than 670,000 total reactions. We found that most mutations (702 of 1036) yielded statistically significant effects on some aspect of catalysis. By systematically and independently varying expression and assay conditions, we determined that 232 of these mutations reduced catalysis by promoting the formation of a long-lived, catalytically inactive misfolded state, whereas none did so through equilibrium unfolding under our assay conditions. Combining these functional measurements with prior mechanistic knowledge allowed us to systematically assess the effect of each mutation. Different groups of residues affected different aspects of function, with residues that affect a particular function forming large, spatially contiguous regions that spanned from the active site up to 20 Å from the active site and to the enzyme surface.
CONCLUSION
HT-MEK has allowed us to uncover functional effects throughout PafA and to identify the catalytic features affected by different groups of residues. Some of these effects are readily rationalized through inspection of structural interconnections to the active site, whereas others were nonobvious, including large distal and surface effects and the discovery of a long-lived misfolded state. These results underscore the need to measure the effects of mutations on multiple kinetic and thermodynamic parameters across multiple reaction conditions and thus the need for this new technology. Because HT-MEK is applicable to any enzyme with a direct or coupled fluorescent readout and provides an in-depth and quantitative analysis of mutant space rapidly and at modest cost, it may be the method of choice to characterize new enzymes. In future applications, HT-MEK can be used to dissect potential evolutionary trajectories, determine the functional consequences of human disease-associated allelic variants, identify surfaces with nascent allosteric potential for rational control of catalysis, and direct the adaptation of natural and designed enzymes for new functions and roles.
Simultaneous expression, purification, and biochemical characterization of enzyme variants in a microfluidic device makes it possible to measure Michaelis-Menten parameters and inhibition constants for more than 1500 variants in days.
Mutational effects across multiple assays reveal an extensive functional architecture in which physically contiguous residue regions extending to the enzyme surface control or alter particular aspects of catalysis.
Abstract
Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.
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