Wednesday, December 31, 2025 12/31/2025

Quantitative, High-throughput Mechanistic Enzymology

Award Number: R01GM064798
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/01/2002
PERIOD OF PERFORMANCE END DATE: 11/30/2027

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Quantitative, High-throughput Mechanistic Enzymology - Project Summary This project will provide data critical to advance our understanding of enzyme sequence/structure/function relationships. We will develop innovative microfluidic tools and techniques and apply them to systematically study enzymes at a high-throughput and quantitative level. In the prior granting period, we developed HT- MEK (High-Throughput Microfluidic Enzyme Kinetics), which enabled recombinant expression, purification, and deep functional characterization of >1500 enzymes in parallel and returns kinetic and thermodynamic constants (e.g. kcat/KM, kcat, KM, Ki) at unprecedented scale. Here, we extend this platform to provide a suite of techniques (HT-MES, High-Throughput Microfluidic Enzyme Stability) capable of quantifying kinetic and thermodynamic stabilities (e.g. ΔGfold, kunfold) with similar throughput. We will also enable site-specific incorporation of noncanonical amino acids to provide the capability to isolate and systematically the impact of particular physicochemical residue properties and to investigate the impacts of post-translational modifications. During the prior granting period, our application of HT-MEK to profile multiple substitutions at each position and multiple functional parameters for the model alkaline phosphatase PafA revealed that residues with similar functional effects on catalysis formed large and spatially contiguous ‘regions’ that extended from the active site to distal surfaces, and that different regions affected different aspects of function. Here, we will systematically apply HT-MEK/S to a variety of new systems to build upon these observations and develop and test new models of how enzymes attain their functions and their observed kinetic and thermodynamic constants. Specifically, we will map functional couplings between residues in PafA via double and multi-mutant cycles and test the degree to which observations from PafA generalize by applying HT-MEK/S to investigate other alkaline phosphatase superfamily members and to acyl phosphatases, which provide ideal enzymes for developing and testing predictive computational models. We will also profile impacts of all nonsynonymous single nucleotide substitutions on folding and catalysis for protein tyrosine phosphatases (PTPs), providing clinically-relevant information about potential health consequences of mutations that can direct development of mechanistically relevant therapies and therapeutics. Finally, we will extend the reach of HT-MEK/S via collaborative projects with the Keedy and Zalatan labs. In all cases, we seek to use high-throughput data to test computational predictions, provide much-needed ground truth data for use by others, and reveal previously unattainable insights into the functional and energetic inner workings of enzymes.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $433,466 )
2026	2026	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	15	12/11/2025	NON-COMPETING CONTINUATION	$433,466
														Subtotal = $433,466

Issue Date FY: 2025 ( Subtotal = $491,753 )
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	14	8/13/2025	NON-COMPETING CONTINUATION	$49,175
2025	2025	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	14	11/26/2024	NON-COMPETING CONTINUATION	$442,578
														Subtotal = $491,753

Issue Date FY: 2024 ( Subtotal = $501,803 )
2024	2024	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	13	1/17/2024	COMPETING CONTINUATION	$501,803
2024	2022	THE LELAND STANFORD JUNIOR UNIVERSITY	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	12	12/5/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $501,803

Issue Date FY: 2022 ( Subtotal = $560,779 )
2022	2022	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	12	8/25/2022	NON-COMPETING CONTINUATION	$560,779
														Subtotal = $560,779

Issue Date FY: 2021 ( Subtotal = $566,535 )
2021	2021	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	11	9/13/2021	NON-COMPETING CONTINUATION	$0
2021	2021	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	11	8/27/2021	NON-COMPETING CONTINUATION	$566,535
														Subtotal = $566,535

Issue Date FY: 2020 ( Subtotal = $569,493 )
2020	2020	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 JANE STANFORD WAY	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	10	9/4/2020	NON-COMPETING CONTINUATION	$569,493
														Subtotal = $569,493

Issue Date FY: 2019 ( Subtotal = $560,999 )
2019	2019	LELAND STANFORD JUNIOR UNIVERSITY, THE	450 SERRA MALL	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	9	9/9/2019	COMPETING CONTINUATION	$560,999
														Subtotal = $560,999

Issue Date FY: 2014 ( Subtotal = $0 )
2014	2012	STANFORD UNIVERSITY	616 SERRA STREET	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	8	1/13/2014	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2012 ( Subtotal = $303,292 )
2012	2012	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	8	1/6/2012	NON-COMPETING CONTINUATION	$272,963
2012	2012	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	8	3/1/2012	NON-COMPETING CONTINUATION	$30,329
														Subtotal = $303,292

Issue Date FY: 2011 ( Subtotal = $301,975 )
2011	2011	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	001	7	7/12/2011	NON-COMPETING CONTINUATION	$27,452
2011	2011	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	7	1/12/2011	NON-COMPETING CONTINUATION	$274,523
														Subtotal = $301,975

Issue Date FY: 2010 ( Subtotal = $301,693 )
2010	2010	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	6	2/25/2010	NON-COMPETING CONTINUATION	$301,693
														Subtotal = $301,693

Issue Date FY: 2009 ( Subtotal = $305,540 )
2009	2009	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	5	3/19/2009	COMPETING CONTINUATION	$305,540
														Subtotal = $305,540

Issue Date FY: 2005 ( Subtotal = $234,928 )
2005	2005	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	4	2/4/2005	NON-COMPETING CONTINUATION	$234,928
														Subtotal = $234,928

Issue Date FY: 2004 ( Subtotal = $234,575 )
2004	2004	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	3	12/4/2003	NON-COMPETING CONTINUATION	$234,575
														Subtotal = $234,575

Issue Date FY: 2003 ( Subtotal = $234,232 )
2003	2003	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	2		NON-COMPETING CONTINUATION	$234,232
														Subtotal = $234,232

Issue Date FY: 2002 ( Subtotal = $233,898 )
2002	2002	STANFORD UNIVERSITY	651 SERRA STREET, ROOM 110	STANFORD	CA	94305	SANTA CLARA	USA	Biomedical Research and Research Training	000	1	2/13/2002	NEW	$233,898
														Subtotal = $233,898

Grand Total All Awards = $5,834,961

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Quantitative, High-throughput Mechanistic Enzymology

Award Number: R01GM064798

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 01/01/2002

PERIOD OF PERFORMANCE END DATE: 11/30/2027

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