This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Wu, G. Articles by Hodge, C. N. Search for Related Content PubMed PubMed Citation Articles by Wu, G. Articles by Hodge, C. N. Social Bookmarking                         What's this?

Determining Appropriate Substrate Conversion for Enzymatic Assays in High-Throughput Screening

Five Prime Therapeutics, San Francisco, CAge.wu{at}fiveprime.com

Yue Yuan

C. Nicholas Hodge

Amphora Discovery Corp., Los Altos, CA

It is generally accepted that the conversion of substrate should be kept at less than 10% of the total substrate used when studying enzyme kinetics. However, 10% or less substrate conversion often will not produce sufficient signal changes required for robust high-throughput screening (HTS). To increase the signal-to-background ratio, HTS is often performed at higher than 10% substrate conversion. Because the consequences of high substrate conversion are poorly understood, the screening results are sometimes questioned by enzymologists. The quality of an assay is judged by the ability to detect an inhibitor under HTS conditions, which depends on the robustness of the primary detection signal (Z factor) and the sensitivity to an inhibitor. The assay sensitivity to an inhibitor is reflected in the observed IC₅₀ value or percent inhibition at a fixed compound concentration when single-point data are collected. The major concern for an enzymatic assay under high substrate conversion is that the sensitivity of the screen may be compromised. Here we derive the relationship between the IC 50 value for a given inhibitor and the percentage of substrate conversion using a first-order kinetic model under conditions that obey Henri-Michaelis-Menten kinetics. The derived theory was further verified experimentally with a cAMP-dependent protein kinase. This model provides guidance for assay developers to choose an appropriate substrate conversion in designing an enzymatic assay, balancing the needs for robust signal and sensitivity to inhibitors.

Key Words: enzyme • substrate • conversion • screening • kinetics

Journal of Biomolecular Screening, Vol. 8, No. 6, 694-700 (2003)
DOI: 10.1177/1087057103260050


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				T. Mori, S. Itami, T. Yanagi, Y. Tatara, M. Takamiya, and T. Uchida Use of a Real-Time Fluorescence Monitoring System for High-Throughput Screening for Prolyl Isomerase Inhibitors J Biomol Screen, April 1, 2009; 14(4): 419 - 424. [Abstract] [PDF]

				Jingsong Yang, R. A. Copeland, and Zhihong Lai Defining Balanced Conditions for Inhibitor Screening Assays That Target Bisubstrate Enzymes J Biomol Screen, February 1, 2009; 14(2): 111 - 120. [Abstract] [PDF]

				C. Marchand, W. A. Lea, A. Jadhav, T. S. Dexheimer, C. P. Austin, J. Inglese, Y. Pommier, and A. Simeonov Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay Mol. Cancer Ther., January 1, 2009; 8(1): 240 - 248. [Abstract] [Full Text] [PDF]

				K. J. Wierenga, K. Lai, P. Buchwald, and M. Tang High-Throughput Screening for Human Galactokinase Inhibitors J Biomol Screen, June 1, 2008; 13(5): 415 - 423. [Abstract] [PDF]

				M. J. Vazquez, S. Ashman, F. Ramon, D. Calvo, A. Bardera, J. J. Martin, M. Rudiger, D. Tew, and J. M. Dominguez Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption J Biomol Screen, February 1, 2006; 11(1): 75 - 81. [Abstract] [PDF]