Enzymes are the primary catalysts of biological transformations and have enormous value in medicine and industry. While decades of research have established that active site residues are essential for catalysis, we do not yet understand in detail the contributions of residues beyond the active site to efficiency and specificity. Of course a folded enzyme is required for catalysis, but beyond folding there is a complex functional interplay of residues throughout an enzyme: allosteric ligand binding and remote mutations alter and enhance catalysis, active site residues coevolve with remote residues and regions, and distal mutations arise in screens and selections for enzymes with enhanced functional properties. Given this inherent complexity, our central premise is that we need tools that extend the power of traditional mechanistic enzymology to systematically investigate residues throughout the entire protein and their interconnectivity. Our central technological innovation delivers the needed tools: High-throughput Microfluidics for Enzyme Kinetics (HT-MEK) and Stability (HT-MES) expresses, purifies, and quantitatively assays 1200 enzyme variants in parallel, rapidly and inexpensively, yielding accurate kinetic and thermodynamic constants for many substrates and ligands over many conditions. With these measurements we will map functional regions and linkages throughout proteins, allowing enzymology to address previously inaccessible challenges in mechanism, evolution, and biology. We first apply these tools to the Alkaline Phosphatase (AP) superfamily member E. meningoseptica PafA, leveraging extensive prior structural, mechanistic, and phylogenetic insights to guide assay development and test previously inaccessible models to deepen our understanding of enzyme catalysis. We will systematically and quantitatively determine kinetic parameters for cognate and promiscuous PafA substrates and affinities for ground and transition-state PafA inhibitors, and we will do so for multiple mutations of every PafA residue; these measurements will provide a comprehensive map of enzyme regions that contribute to specific components of catalytic function. Next, we will use multi-mutant cycles to determine the energetic and functional linkage of these regions to active site residues and specific catalytic features, as well as the connections within and between these regions. Extension to multiple AP superfamily members across evolutionary distances will identify the range and limits of generality of functional maps and identify the alterations that rewire functional connectivity. Expanding this approach to other targets, some of which are explored herein, will address fundamental and practical problems of broad interest. This carefully-reasoned, stepwise approach will usher in a new era of enzymology, in which the acquisition of multidimensional functional maps of enzymes addresses new questions in mechanism, evolution, and biology, and in which enzymology impacts biomedicine and engineering in new ways.

Public Health Relevance

Enzymes are the catalytic machines that drive biological reactions, and the ability to design and engineer them for specific and efficient catalysis of new reactions would have profound impacts across industry and medicine. To date, designed enzymes have yielded catalytic enhancements orders of magnitude less than those of natural enzymes, and we lack experimental tools to systematically and quantitatively investigate how residues in naturally-occurring enzymes proximal and distal to the active site contribute to catalysis. We present a suite of new high-throughput tools with the precision and information content of traditional enzymology, yet which require only a small fraction of the time and cost; with these tools we will map functional regions and linkages throughout entire proteins, allowing enzymology to address previously inaccessible challenges in mechanism, evolution, and biology and precipitating new applications in biomedicine and engineering.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM064798-09
Application #
9819655
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
2002-01-01
Project End
2023-08-31
Budget Start
2019-09-11
Budget End
2020-08-31
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem et al. (2017) Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution. J Biol Chem 292:20960-20974
Peck, Ariana; Sunden, Fanny; Andrews, Logan D et al. (2016) Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase. J Mol Biol 428:2758-68
Sunden, Fanny; Peck, Ariana; Salzman, Julia et al. (2015) Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site. Elife 4:
Shah, Niket; Colbert, Karen N; Enos, Michael D et al. (2015) Three ?SNAP and 10 ATP molecules are used in SNARE complex disassembly by N-ethylmaleimide-sensitive factor (NSF). J Biol Chem 290:2175-88
Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel (2014) Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases. Biochemistry 53:6811-9
Khosla, Chaitan; Herschlag, Daniel; Cane, David E et al. (2014) Assembly line polyketide synthases: mechanistic insights and unsolved problems. Biochemistry 53:2875-83
Andrews, Logan D; Fenn, Tim D; Herschlag, Daniel (2013) Ground state destabilization by anionic nucleophiles contributes to the activity of phosphoryl transfer enzymes. PLoS Biol 11:e1001599
Herschlag, Daniel; Natarajan, Aditya (2013) Fundamental challenges in mechanistic enzymology: progress toward understanding the rate enhancements of enzymes. Biochemistry 52:2050-67
Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel (2013) Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase. Biochemistry 52:9167-76
Althoff, Eric A; Wang, Ling; Jiang, Lin et al. (2012) Robust design and optimization of retroaldol enzymes. Protein Sci 21:717-26

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