Enzymes are used to facilitate a variety of industrial processes due to their unique biochemical capabilities, and because of the critical role they play in many biological processes, they are also a popular drug target. One common approach for understanding the function of enzymes is site-directed mutagenesis, in which the genetic blueprint for a specific part of an enzyme is altered to see how the mutation affects its behavior, but this process can be very time- and resource-intensive. This can limit researchers’ focus to the active site of an enzyme, which may not provide a full picture of how other sites play a role in its function. Researchers from Stanford University developed a new tool for investigating many more areas of an enzyme in a much shorter period of time by combining two time-saving technologies: microfluidics and cell-free protein synthesis.
The new technology, called High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), involves running more than a thousand mutagenesis experiments in separate nanoliter-sized channels within a single device, allowing researchers to explore many more enzyme sites at once. The tiny microfluidic channels greatly reduce the physical space and volume of resources needed to complete many experiments, and the use of cell-free protein synthesis removes several more steps from the process. Combining these two technologies significantly boosts throughput while providing a much more complete picture of the enzyme being studied. The researchers further streamlined the process by using a printer to deposit the thousand-plus different microscopic bits of altered DNA onto a slide before aligning them with the channels containing the mix of chemicals needed for cell-free synthesis.
The research team used HT-MEK to investigate the enzyme PafA and found that mutations of amino acid residues well beyond the active site affected the enzyme’s ability to catalyze chemical reactions. The study also revealed that a surprising number of mutations caused PafA to misfold into an inactive state, many of which would be difficult to identify through more limited mutagenesis experiments. This research was published in Science.
“If you’re an enzymologist trying to learn about a new enzyme and you have the opportunity to look at 5 or 10 mutations over six months or 100 or 1,000 mutants of your enzyme over the same period, which would you choose? This is a tool that has the potential to supplant traditional methods for an entire community,” said study co-leader Dan Herschlag.
This high-throughput method could be especially useful for identifying allosteric drug targets. By understanding how parts of an enzyme other than the active site contribute to the enzyme’s catalytic ability, pharmaceutical researchers can develop drugs that are more selective and result in fewer side effects. The method can also be used to better engineer enzymes for industrial processes and create sustainable solutions, such as new enzymes that can break down plastics.
Photo: HT-MEK -- short for High-Throughput Microfluidic Enzyme Kinetics -- combines microfluidics and cell-free protein synthesis technologies to dramatically speed up the study of enzymes. Credit: Daniel Mokhtari