Elsevier

Methods

Volume 36, Issue 4, August 2005, Pages 321-331
Methods

Assays for mechanistic investigations of protein/histone acetyltransferases

https://doi.org/10.1016/j.ymeth.2005.03.002Get rights and content

Abstract

Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or α-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

Introduction

At least five unique histone acetyltransferase (HAT) gene families have been described [1], [2] and in all cases, acetyl-CoA functions as the acetyl donor, transferring this group to the epsilon amino group of lysine side chains within histone proteins. Although capable of acetylating histones, other non-histone proteins have been shown to be in vivo and in vitro targets of several acetyltransferases [1], [3], [4]. Given the prevalence of both histone and non-histone protein acetylation, some of these HATs may be more appropriately referred to as protein acetyltransferases (PATs). The importance of histone/protein acetyltransferases in a variety of biological processes, and the reported diversity in their target substrates and structures, addresses the need to understand the molecular mechanism of each gene family. Acetyl-CoA-dependent acetyltransferases are known to utilize one of two catalytic mechanisms, which are depicted in Fig. 1. One mechanism involves the formation of an acetylated enzyme intermediate after binding and reaction with acetyl-CoA (Fig. 1, upper panel). The product CoA is released, protein substrate binds, and reacts with the intermediate to generate the final acetylated protein product. With a ternary complex mechanism (Fig. 1, lower panel), both substrates must bind to form a ternary complex allowing the lysine to directly attack the bound acetyl-CoA, without the formation of a covalent enzyme intermediate. Considerable evidence exists for Gcn5/PCAF HATs [5], [6], [7], [8] following a ternary complex mechanism (sequential), while Esa1 related HATs [9] may utilize an enzyme intermediate mechanism (ping pong). Here we describe quantitative methods to measure acetyltransferase activity, where the resulting kinetic data are useful in determining molecular mechanisms and in probing substrate preferences among diverse acetyltransferases. Although we will focus our discussion on HATs, the general concepts are applicable to acetyl-CoA-dependent acetyltransferases that target non-histone proteins [1].

Section snippets

General assay considerations

The assays described in this paper are useful in generating steady-state rate constants, which can provide mechanistic information on how the enzyme carries out catalysis, and can be used to compare the efficiencies of different HATs, to probe the function of accessory proteins, and to compare relative substrate preferences. More detailed discussions on both the practical and theoretical aspects of enzyme assays can be found in [10], [11], [12]. Simple substrate saturation curves yield data on V

Representative HAT assays

A typical filter-binding assay uses phosphocellulose discs to preferentially bind acetylated histones [5], [13], [14], [15], [16]. Unincorporated radioactivity from radiolabeled acetyl-CoA is removed from the discs with extensive washing. The bound radioactivity is measured by liquid scintillation counting and converted to rate data. This assay method is the most sensitive of the HAT assays, allowing one to discern between subtle differences in rates while consuming very little enzyme. However,

Filter-binding assays

First, we will describe the materials and setup required for the filter-binding assay, as well as describe how to measure product formed and how to determine rate data from a time course or progress curve.

Continuous enzyme-coupled assay

This basic enzyme-coupled assay was first developed a few years ago [19]. Here, we will describe the general methods and its utilization in a high-throughput assay.

Optimization of the enzyme-coupled assay

The coupled reaction components of this assay should be optimized to ensure that the coupled reaction rate does not limit the HAT reaction. The conditions described in the next section were optimized by Kim et al. [19] for yGCN5 and a peptide substrate. Optimization of the assay conditions is necessary for each enzyme and peptide substrate to avoid limiting the PAT/HAT reaction by the coupled reaction. To examine this, pyruvate (or α-ketoglutarate), PDH (or α-ketoglutarate dehydrogenase), and

Generating substrate saturation curves from initial velocities

Both the assays detailed in this paper can be used to generate substrate saturation curves, and subsequently, to determine the steady-state kinetic parameters (Vmax, Km, and V/K). A saturation curve for an acetyltransferase is performed by holding one substrate in excess (∼10 times the Km) while varying the concentration of the other over a range (⩾10×-fold) that straddles the Km, and initial reaction velocities are measured. These rates are plotted versus the concentration of the varied

Generation of progress curves and use of the integrated Michaelis–Menten equation

A limitation of most methods discussed here is the amount of substrate required to generate saturation curves. This is especially true for the most physiologically relevant HAT substrates, like nucleosomes or nucleosomal arrays. These native HAT substrates, whether purified from natural sources or by recombinant reconstitution, cannot always be obtained in quantities necessary to generate a traditional saturation curve. We have developed an approach using the filter-binding assay and the

Use of quantitative assays to probe catalytic mechanism and substrate preferences

The assays described in this paper are suitable for mechanistic studies on a wide range of protein and histone acetyltransferases. These assays can be used to distinguish between the two mechanisms (ping pong versus sequential) depicted in Fig. 1, to probe substrate specificity and to compare catalytic efficiencies among diverse HAT complexes. A bi-substrate kinetic analysis (both substrates are varied) can provide strong evidence in favor of one of the catalytic mechanisms (Fig. 1). The data

Acknowledgments

We thank Dr. Song Tan from Penn State University for the expression plasmids of picNuA4 and for providing nucleosome core particles, which were used as an example of the methods in Fig. 3, Fig. 5.

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    This work was supported in part by Grant GM065386 from the NIH and by Grant RSG-01-029-01-CNE from the American Cancer Society.

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