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Transition state analysis

MutY TSEnzymes catalyze reactions by binding to and stabilizing the transition state (TS) (learn about TSs here). They bind TSs tighter than reactants or products by up to 1020-fold. Developing a deep understanding of catalysis, and designing effective new enzyme inhibitors therefore requires understanding the TS structure. The challenge in studying TSs is that they exist for ~100 fs, and their concentration is effectively zero.

The only way to experimentally determine TS structures is measuring kinetic isotope effects (KIEs) (learn about KIEs here). Only a handful of labs in the world are able to perform TS analysis using KIEs.

Published work

We determined the TS structures for:
• the antibiotic target AroA (link) and the corresponding non-enzymatic reaction (link)
• the DNA repair enzyme MutY(link), and for the corresponding acid-catalyzed reaction (link)
• α- and β-glucosidase-catalyzed hydrolysis of α- and β-methyl glucoside, and the corresponding acid-catalyzed reactions (link)


Current projects

We are currently performing TS analysis on several enzymes.

MGAM and SI — targets for diabetes control

maltoseHuman maltase/glucoamylase (MGAM) and sucrase/isomaltase (SI) are α-glucosidases — enzymes that hydrolyse glycosidic bonds in order to digest complex carbohydrates. They release glucose and fructose into the bloodstream, which leads to postprandial hyperglycemia in diabetics. Several α-glucosidase inhibitors (acarbose, miglitol, voglibose) are used to slow glucose release into the bloodstream, and thereby help to control diabetes. These drugs are not optimal; the most potent inhibitors cause serious side effects, and those with fewer side effects are not very effective inhibitors.

We are performing TS analysis on these enzymes in order to better understand the details of catalysis, which will ultimately become a blueprint for the design of better α-glucosidase inhibitors.

α-Carboxyketose synthases

In addition to creating new inhibitors of the antimicrobial targets, the α-carboxyketose synthases, we are interested in determining their TS structures as a probe of mechanism, and to create a new blueprint for inhibitor design.


AlkA — a DNA repair enzyme

AlkA rxnOur DNA is constantly being damaged; every 5 ps a DNA base gets oxidized, deaminated or alkylated somewhere in our body. If this damage is not repaired, mutations will accumulate, leading to a loss of genetic information, cell damage, and ultimately cancer.

Alkylation accelerates nucleoside bond hydrolysis by 5 × 105-fold, accelerating DNA breakdown. AlkA recognizes damaged bases and removes them, the first step in base excision repair. It has a broad specificity, recognizing both alkylated and deaminated purine bases. Aside from the purine ring, there are no common features in the base portion of its substrates.  One of the major challenges in understanding AlkA catalysis is understanding how an enzyme can recognize and hydrolyze a variety of damaged DNA bases without hydrolyzing the millions-fold excess of normal DNA. We are performing TS analysis on the AlkA reaction in order to understand how it achieves broad specificity and while avoiding damaging normal DNA.

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