Just because an enzyme has an active site doesn’t mean the rest of the enzyme should be disregarded, as though it were so much dead weight. It influences enzyme function, if only by keeping the enzyme from unraveling and losing its function altogether. The rest of the enzyme may also fine-tune enzyme function in ways that are rather more subtle—too subtle, alas, to be determined without the patient study of enzyme variants, that is, versions of the enzyme that incorporate one or more mutations. Because studying how mutations affect enzyme function is such a painstaking business, the mutations that are assessed tend to be close to—you guessed it—the active site.

To broaden enzyme function studies so they may take in the whole of enzymes, scientists at Stanford University developed High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1,500 enzyme variants per experiment. HT-MEK, the Stanford team asserted, can compress years of work into just a few weeks by enabling thousands of enzyme experiments to be performed simultaneously.

The team—led by Polly Fordyce, PhD, an assistant professor of bioengineering and of genetics at Stanford University, and Dan Herschlag, PhD, a professor of biochemistry at Stanford’s School of Medicine—applied HT-MEK to a well-studied enzyme called PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium). The study’s results appeared in Science, in a paper titled, “Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.”

“For 1,036 mutants of the alkaline phosphatase PafA, we performed more than 670,000 reactions and determined more than 5,000 kinetic and physical constants for multiple substrates and inhibitors,” the article’s authors reported. “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—short for High-Throughput Microfluidic Enzyme Kinetics—combines microfluidics and cell-free protein synthesis technologies to dramatically speed up the study of enzymes. [Daniel Mokhtari, Stamford University]
HT-MEK combines two existing technologies to rapidly speed up enzyme analysis. The first is microfluidics, which involves molding polymer chips to create microscopic channels for the precise manipulation of fluids. “Microfluidics shrinks the physical space to do these fluidic experiments in the same way that integrated circuits reduced the real estate needed for computing,” said Fordyce. “In enzymology, we are still doing things in these giant liter-sized flasks. Everything is a huge volume, and we can’t do many things at once.”

The second is cell-free protein synthesis, a technology that takes only those crucial pieces of biological machinery required for protein production and combines them into a soupy 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 nanoliter-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 liter 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 constitute the enzyme. In this way, it’s possible to systematically study how different modifications to an enzyme affects its folding, catalytic ability, and ability to bind small molecules and other proteins.

By allowing scientists to deeply probe beyond the small active site of an enzyme where substrate binding occurs, HT-MEK could reveal clues about how even the most distant parts of enzymes work together to achieve their remarkable reactivity.

Essentially, HK-MEK could help scientists avoid the Streetlight Effect, a type of observational bias that is best described metaphorically: a drunkard looks for lost keys beneath a streetlight not because it is where they’re most likely to be found, but because it is, the drunkard says, “where the light is.”

“It’s like we’re now taking a flashlight and instead of just shining it on the active site, we’re shining it over the entire enzyme,” Fordyce explained. “When we did this, we saw a lot of things we didn’t expect.”

For example, the scientists found that mutations well beyond the active site affected PafA’s ability to catalyze chemical reactions—indeed, most of the amino acids, or “residues,” making up the enzyme had effects. The scientists also discovered that a surprising number of mutations caused PafA to misfold into an alternate state that was unable to perform catalysis.

“Combined with recent advances in gene synthesis, HT-MEK can rapidly functionally characterize metagenomic variants, providing a critically needed dimension to phylogenetic analyses,” the authors of the Science article concluded. “In medicine, we anticipate that HT-MEK will rapidly determine the functional effects of human enzyme allelic variants of unknown relevance identified from sequence data and systematically identify candidate allosteric surfaces within currently ‘undruggable’ therapeutic target enzymes. We anticipate HT-MEK contributing to these and still more areas of basic and applied biology, medicine, and engineering.”