Scientists understand that transformations that couldn’t historically be explained by conventional chemistry or physics alone are in fact powered by enzymes – protein molecules comprised of chains of amino acids that act to speed up, or catalyse, the conversion of one kind of molecule (substrates) into another (products).
While more is now known about enzymes, including their structures and the chemical groups they use to facilitate reactions, the details surrounding how their forms connect to their functions, and how they achieve biochemical reactions with such speed and specificity, are still not well understood. Polly Fordyce, an Assistant Professor of Bioengineering and of Genetics at Stanford University said: “A chemical reaction that would take longer than the lifetime of the universe to happen on its own can occur in seconds with the aid of enzymes.” A new technique, developed by Fordyce and her colleagues at Stanford and published this week in Science, could help change that.
Dubbed HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), the technique can compress years of work into a few weeks by enabling thousands of enzyme experiments to be performed simultaneously. “Limits in our ability to do enough experiments have prevented us from truly dissecting and understanding enzymes,” said study co-leader Dan Herschlag, a Professor of Biochemistry at Stanford’s School of Medicine.
HT-MEK is designed to replace the process for purifying enzymes that has traditionally involved engineering bacteria to produce a particular enzyme, growing them in large beakers, bursting open the microbes and then isolating the enzyme of interest from all the other unwanted cellular components. To piece together how an enzyme works, scientists introduce intentional mistakes into its DNA blueprint and then analyse how these mutations affect catalysis. This process is expensive and time consuming, however, so researchers have mostly limited their scientific investigations to the active sites of enzymes.
The technology behind HT-MEK was developed and refined over six years through a partnership between the labs of Fordyce and Herschlag. It combines two existing technologies to rapidly speed up enzyme analysis. The first is microfluidics, which involves moulding polymer chips to create microscopic channels for the precise manipulation of fluids. The second is cell-free protein synthesis, a technology that takes the pieces of biological machinery required for protein production and combines them into an extract that can be used to create enzymes synthetically, without requiring living cells to serve as incubators.
“We’ve automated it so that we can use printers to deposit microscopic spots of synthetic DNA coding for the enzyme that we want onto a slide and then align nanolitre-sized chambers filled with the protein starter mix over the spots,” Fordyce explained.
Because each tiny chamber contains only a thousandth of a millionth of a litre of material, the scientists can engineer thousands of variants of an enzyme in a single device and study them in parallel. By tweaking the DNA instructions in each chamber, they can modify the chains of amino acid molecules that comprise the enzyme. In this way, it is possible to systematically study how different modifications to an enzyme affects its folding, catalytic ability and ability to bind small molecules and other proteins.
When the team applied their technique to a well-studied enzyme called PafA, they found that mutations well beyond the active site affected its ability to catalyse chemical reactions. Most of the amino acids, or “residues,” making up the enzyme had effects. “Biochemists have known for decades that misfolding can occur but it’s been extremely difficult to identify these cases and even more difficult to quantitatively estimate the amount of this misfolded stuff,” said study co-first author Craig Markin, a research scientist with joint appointments in the Fordyce and Herschlag labs.
If widely adopted, researchers say that HT-MEK will not only improve the basic understanding of enzyme function, but also catalyse advances in medicine and industry. It could also accelerate an approach to drug development called allosteric targeting, which aims to increase drug specificity by targeting beyond an enzyme’s active site. Enzymes are popular pharmaceutical targets because of the key role they play in biological processes. But some are considered “undruggable” because they belong to families of related enzymes that share the same or very similar active sites, and targeting them can lead to side effects.
The sheer amount of data that HT-MEK is expected to generate will reportedly benefit computational approaches and machine learning algorithms, like the Google-funded AlphaFold project, designed to deduce an enzyme’s complicated 3D shape from its amino acid sequence alone.
Much further down the road, it is suggested that HT-MEK may allow scientists to reverse-engineer enzymes and design bespoke varieties of their own. Fordyce said, in way of example: “We would love to create enzymes that can degrade plastics into nontoxic and harmless pieces. If it were really true that the only part of an enzyme that matters is its active site, then we’d be able to do that and more already. Many people have tried and failed, and it’s thought that one reason why we can’t is because the rest of the enzyme is important for getting the active site in just the right shape and to wiggle in just the right way.”