Enzyme Mechanism and Evolution
Catalysis is central to biology and is carried out by enzymes –molecular machines that provide the tremendous rate enhancements and extraordinary specificities needed for the functioning of all organisms. Enormous progress has been made in understanding enzyme mechanisms over the past decades; nevertheless, fundamental questions and challenges remain. In particular, whereas the chemical mechanisms of most enzymes are known, unraveling the underlying energetic mechanisms is an exciting current challenge. This understanding is fundamental to modern-day biology, and how it evolved and continues to evolve, and will contribute to better design of novel artificial enzymes and enzyme inhibitors and drugs.
There have been astounding advances in the understanding of enzyme mechanism over the past decades. Nevertheless, fundamental questions remain, and indeed, much of the previous work has helped to bring these critical questions into focus.
We focus on enzymes that allow us to ask directed and incisive questions about how enzymes achieve their enormous rate enhancements and exquisite specificities, how these enzymes have evolved, and how we can achieve sufficient understanding to engineer new enzymes for health and engineering.
Ketosteroid Isomerase: Investigating fundamental chemical and physical principles of enzyme catalysis
KSI is a remarkably tractable enzyme. There is an established kinetic and thermodynamic reaction framework. KSI has available transition state analogs, high-resolution crystal structures, can be made semi-synthetically with unnatural amino acids incorporated, and its small size and high solubility allow application of multiple spectroscopic probes, including NMR and vibrational spectroscopy.
KSI catalyzes a double bond isomerization in steroid substrates and the reaction is accelerated by twelve orders of magnitude as compared to the uncatalyzed solution reaction. A fundamental challenge is to understand the unique features within the enzyme that enable this rate enhancement. Key catalytic strategies identified thus far in the active site are the general base and oxyanion hole, as well as a substrate binding pocket. While the general base transfers the proton on the steroid substrate, the oxyanion hole stabilizes the reaction transition states and intermediate by donating hydrogen bonds to the incipient oxyanion. These catalytic residues are positioned through multiple interactions and are embedded within the enzyme’s scaffold.
There have been many proposals about the structure and energetics of hydrogen bonds. The oxyanion hole of KSI has provided us the most powerful system to test multiple hypotheses and establish quantitative understanding. This work is ongoing.
There has been much discussion about roles of dynamics in enzymatic catalysis. Nevertheless, it has been difficult to define the important questions and thus to address them clearly and systematically.
KSI provides an important perspective to gain insights into hydrogen bonds and the positioning of catalytic groups, as is needed for catalysis, and the conformational mobility that remains in the active site and throughout an enzyme. We are using state-of-the-art NMR dynamic and room temperature crystallography to systematically explore the interplay of dynamics, energetics, and catalysis via a series of KSI ground and transition state analog complexes, as well as other chemical, biochemical, and biophysical approaches.
Catalytic Promiscuity and the Alkaline Phosphatase Superfamily
Whereas enzymes are widely renowned for the exquisite specificity, we showed that enzymes also have low levels of catalysis for alternative reactions, an observation we described as ‘catalytic promiscuity’.
Catalytic promiscuity has been a key driver in the evolution of new enzymes, providing a head start and selective advantage for enzymes already possessing a low level of advantageous activity.
Catalytic promiscuity provides a powerful tool for addressing evolutionary and mechanistic questions.
We are exploiting catalytic promiscuity across an enzyme superfamily 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. Because the substrates vary 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 several members of this superfamily.
We have identified and distinguished the common and unique features of AP superfamily members, some that provide a catalytic boost for all reactions and some that allow specialization for a particular cognate reaction.
We have carried out the most in-depth functional dissection of an active site, determining the energetic connectivity between five active site residues and identifying three interconnected functional units. Work continues to expand and deepen these interconnections.
Our work has implications for the fundamental catalytic function of enzymes, the design of new enzymes, and understanding how enzymes have evolved.
We are now working with the Fordyce Lab to greatly deepen and broaden this work. We are using a novel microfluidic platform to quantitatively study thousands of enzyme variants in parallel, thereby allowing us to investigate functional and structural coupling throughout an entire enzyme for the first time. In addition, our results to date have lead to several hypotheses about active site connectivity and function that we are in the processing of testing. Our functional and kinetic work is tightly tied to x-ray crystallographic structure determination.
1. Herschlag, D., Natarajan, A. (2013) Biochemistry 52, 2050-2067. Fundamental Challenges in Mechanistic Enzymology: Progress toward understanding the Rate Enhancements of Enzymes. PMCID: PMC3744632 (Medline) (PDF File)
2. Lassila, J.K., Zalatan, J.G., Herschlag, D. (2011) Annu. Rev. Biochem. 80, 669-702. Biological Phosphoryl Transfer Reactions: Understanding Mechanism and Catalysis. PMCID: PMC3418923. (Medline) (PDF File) (Supporting Info)
3. Zalatan, J.G., Herschlag, D. (2009) Nature Chem. Biol. 5, 516-520. The Far Reaches of Enzymology. PMID: 19620986. (Medline) (PDF File)
4. Kraut, D.A., Carroll, K.S., Herschlag, D. (2003) Annu. Rev. Biochem. 72, 517-571. Challenges in Enzyme Mechanism and Energetics. PMID: 12704087. (Medline) (PDF File)
5. O'Brien, P., Herschlag, D. (1999) Chemistry and Biology 6, R91-R105. Catalytic Promiscuity and the Evolution of New Enzymatic Activities. PMID: 10099128. (Medline) (PDF File)
6. Narlikar, G.J., Herschlag, D. (1997) Annu. Rev. Biochem. 66, 19-59. Mechanistic Aspects of Enzymatic Catalysis: Lessons from Comparison of RNA and Protein Enzymes. PMID: 9242901. (Medline) (PDF File)
1. Lassila, J.K., Baker, D., Herschlag, D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 4937-4942. Origins of Catalysis by Computationally Designed Retroaldolase Enzymes. PMCID: PMC2841948. (Medline) (PDF File) (Supporting Info)
See also: Faculty of 1000 Biology
2. Shan, S., Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14474-14479. The Change in Hydrogen Bond Strength Accompanying Charge Rearrangement: Implications for Enzymatic Catalysis. PMCID: PMC26157. (Medline) (PDF File)
3. Shan, S., Loh, S., Herschlag, D. (1996) Science 272, 97-101. The Energetics of Hydrogen Bonds in Model Systems. Implications for Enzymatic Catalysis. PMID: 8600542. (Medline) (PDF File)
Some leading papers from the lab on KSI:
1. Lamba, V., Yabukarski, F., Pinney, M., Herschlag, D. (2016) J. Am. Chem. Soc. In Press. Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase.
2. Sigala, P., Ruben, E., Liu, C., Piccoli, P., Hohenstein, E., Martinez, T., Schultz, A., Herschlag, D. (2015) J. Am. Chem. Soc. 137, 5730-5740. Determination of Hydrogen Bond Structure in Water Versus Aprotic Environments to Test the Relationship Between Length and Stability. PMID: 25871450 (Medline) (PDF File) (Supporting Info)
3. Natarajan, A., Schwans, J.P., Herschlag, D. (2014) J. Am. Chem. Soc. 136, 7643-7654. Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site. pmcid: pmc4046884 (Medline) (PDF File) (Supplementary Info)
4. Schwans, J.P., Sunden, F., Lassila, J.K., Gonzalez, A., Tsai, Y., Herschlag, D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 11308-11313. Use of Anion Aromatic Interactions to Position the General Base in the Ketosteroid Isomerase Active Site. PMCID: PMC3710852. (Medline) (PDF File) (Supporting Info)
See also: Faculty of 1000 Biology, July 2013
5. Schwans, J., Sunden, F., Gonzalez, A., Tsai, Y., Herschlag, D. (2011) J. Am. Chem. Soc. 133, 20052-20055. Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase. PMCID: PMC3241876. (Medline) (PDF File) (Supporting Info)
See also: Schwans, J., Sunden, F., Gonzalez, A., Tsai, Y., Herschlag, D. (2016) J. Am. Chem. Soc. Correction to "Evaluating the Catalytic Contribution from the
Oxyanion Hole in Ketosteroid Isomerase." (Medline) (PDF File) (Supporting Info)
6. Sigala, P.A., Tsuchida, M.A., Herschlag, D. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 9232-9237. Hydrogen Bond Dynamics in the Active Site of Photoactive Yellow Protein. PMCID: PMC2695108. (Medline) (PDF File) (Supporting Info)
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7. Sigala, P., Kraut, D., Caaveiro, J., Pybus, B., Ruben, E., Ringe, D., Petsko, G., Herschlag, D. (2008) J. Am. Chem. Soc. 130, 13696-13708. Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole. PMCID: PMC2700827. (Medline) (PDF File) (Supporting Info)
See also: Nature 2008, 456, 45-47.
8. Sigala, P.A., Farfarman, A.T., Bogard, P.E., Boxer, S.G., Herschlag, D. (2007) J. Am. Chem. Soc. 129, 12104-12105. Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site? PMCID: PMC3171184. (Medline) (PDF File) (Supporting Info)
See also: Faculty of 1000 Biology, October 2007
9. Kraut, D.A., Sigala, P.A., Pybus, B., Liu, C.W., Ringe, D., Petsko, G.A., Herschlag, D. (2006) PLoS Biology 4, e99. Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole. PMCID: PMC1413570. (Medline) (PDF File) (Supporting Info)
Some leading papers from the lab on catalytic promiscuity and phosphoryl transfer:
1. Sunden, F., Peck, A., Salzman, J., Ressl, S., Herschlag, D. (2015) eLife 4, e06181. Extensive Site-directed Mutagenesis Reveals Interconnected Functional Units in the Alkaline Phosphatase Active Site. PMCID: PMC4438272 (Medline) (PDF File) (Supporting Info)
See also: Protein engineering: Beating the odds, December 2015
2. Andrews, L., Fenn, T., Herschlag, D. (2013) PLoS Biology 11, 1-18. Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes. PMCID: PMC3699461 (Medline) (PDF File)
See also: Faculty of 1000 Biology, July 2013
3. Zalatan, J., Fenn, T.D., Herschlag, D. (2008) J. Mol. Biol. 384, 1174-1189. Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion. PMCID: PMC2622731. (Medline) (PDF File) (Supporting Info)
4. Zalatan, J.G., Fenn, T.D., Brunger, A.T., Herschlag, D. (2006) Biochemistry 45, 9788-9803. Structural and Functional Comparisons of Nucleotide Pyrophosphatase/Phosphodiesterase and Alkaline Phosphatase: Implications for Mechanism and Evolution. PMID: 1689310. (Medline) (PDF File) (Supporting Info)
5. O'Brien, P., Herschlag, D. (2002) Biochemistry 41, 3207-3225. Alkaline Phosphatase Revisited: The Hydrolysis of Alkyl Phosphates. PMID: 1186460. (Medline) (PDF File) (Supporting Info)
6. Cheng, H., Nikolic-Hughes, I., Wang, J.H., Deng, H., O'Brien, P.J., Wu, L., Zhang, Z.Y., Herschlag, D., Callender, R. (2002) J. Am. Chem. Soc. 124, 11295-11306. Environmental Effects on Phosphoryl Group Bonding Probed by Vibrational Spectroscopy: Implications for Understanding Phosphoryl Transfer and Enzymatic Catalysis. PMID: 12236744. (Medline) (PDF File)
7. O'Brien, P., Herschlag, D. (1999) Chemistry and Biology 6, R91-R105. Catalytic Promiscuity and the Evolution of New Enzymatic Activities. PMID: 10099128. (Medline) (PDF File)
8. Maegley, K.A., Admiraal, S.J., Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8160-8166. Ras-catalyzed Hydrolysis of GTP: Insights from Model Studies. PMID: 8710841. (Medline) (PDF File)
9. Hollfelder, F., Herschlag, D. (1995) Biochemistry 34, 12255-12264. The Nature of the Transition State for Enzyme-catalyzed Phosphoryl Transfer. Hydrolysis of O-Arylphosphorothioates by Alkaline Phosphatase. PMID: 7547968. (Medline) (PDF File)
10. Admiraal, S.J., Herschlag, D. (1995) Chemistry and Biology 2, 729-739. Mapping the Transition State for ATP Hydrolysis. Implications for Enzymatic Catalysis. PMID: 9383480. (Medline) (PDF File)
11. Herschlag, D. (1994) J. Am. Chem. Soc. 116, 11631-11635. Ribonuclease Revisited: Catalysis Via the Classical General Acid-Base Mechanism or a Triester-like Mechanism? (PDF File)