Ongoing Research Topics
The overarching goal of the Herschlag Lab is to understand the fundamental behavior of RNA and proteins and, in turn, how these behaviors determine and impact biological catalysis and biology. The lab takes an interdisciplinary approach, spanning and integrating physics, chemistry and biology through fruitful interactions with collaborators and a wide range of techniques are employed.
Ketosteroid Isomerase Catalysis
Enzyme Promiscuity, Evolution, and Phosphoryl Transfer
In recent years it has been recognized that proteins in the same superfamily –i.e.,
evolutionarily related proteins that possess similar structural motifs– often have low levels of activity toward reactions catalyzed by other members within the superfamily. This property, coined ‘catalytic promiscuity’, provides a powerful tool for addressing both evolutionary and mechanistic questions. We are exploiting this property of enzyme superfamilies to gain an understanding of the fundamental underpinnings of catalysis. Unlike traditional site-directed mutagenesis experiments that are often limited to studying a single reaction catalyzed by an individual enzyme, we are using a comparative approach to ask not simply what the consequence is of removing a particular side chain, but how that removal and change in the side chain affects normal and promiscuous reactions. Conversely, we can also ask how changes in an analogous residue in another member of the superfamily affect its cognate and promiscuous activity. Because the substrates varying in their charge, geometry, and transition state natures, the observed effects tell us more precisely about the catalytic roles of particular interactions and how these enzymes have become optimized over time for catalysis of their cognate reactions.
The Alkaline Phosphatase (AP) superfamily provides a powerful system in which to address these questions and also allows us to address the important and widespread biological reactions that involve phosphoryl transfer. Members of the AP superfamily catalyze a range of reactions including phosphoryl and sulfuryl transfer reactions. We currently focus work on studying two members of this superfamily, alkaline phosphatase (AP) and nucleotide pyrophosphatase/phosphodiesterase (NPP). AP preferentially catalyzes phosphate monoester hydrolysis reactions, whereas NPP is a proficient diesterase. Both enzymes have promiscuous activities toward each other’s reactions. Although these enzymes catalyze different reactions with a specificity difference of >1013-fold, AP and NPP share an indistinguishable Zn2+ bimetallo site. We use a variety of structural and functional probes to elucidate the origins of this specificity difference. These approaches include steady stateand pre-steady state kinetic comparisons of cognate and non-cognate substrate reactions with wild type and mutant enzymes to determine rate enhancements and to dissect catalysis; structural comparisons between homologous enzymes to guide and interpret site-directed mutagenesis; X-ray crystallography and EXAFS to compare structures of homologous enzymes and to determine the structural consequences of mutations; vibrational spectroscopy to assess the nature of bound ligands; binding of substrates, inhibitors, and transition state analogs to wild type and mutant enzymes to further compare homologous enzymes and determine the energetic consequences of mutations; and linear free energy relationships and heavy atom isotope effects to obtain information about the reactions’ transition states and their active site interactions.
Some leading papers from the lab in the areas of catalytic promiscuity and phoshoryl transfer are: