Ronald G. Duggleby

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

Summary.The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

A nonlinear regression program for small computers

A BASIC computer program for performing weighted nonlinear regression is described and a listing of the program is given. The program, which is small and simple to use, has been designed to be run by users with little knowledge of mathematics or computers. Robust methods of analysis are described which may be applied to data in which experimental errors are not normally distributed, and the program incorporates one such method. It is shown that the program is useful for the analysis of data conforming to the Michaelis-Menten equation, a single exponential, and to binding equations, and other applications are discussed.

Regression analysis of nonlinear Arrhenius plots: An empirical model and a computer program

The rates of most physical, chemical and biological processes vary with temperature and numerous instances have been reported in which Arrhenius plots of the experimental data appear to consist of two straight line segments joined by a relatively sharp break. An empirical model, based on a general hyperbola, is shown to be applicable to such systems; a nonlinear regression program is described which facilitates fitting of this function to experimental data which show either broad or sharp transitions. The program yields best estimates and standard errors of the temperature and ordinate value at the transition, and the two activation energies.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimers disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Å resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K i of 3.3 nm, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.

Structure of a human carcinogen-converting enzyme, SULT1A1. Structural and kinetic implications of substrate inhibition.

Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, metabolizes and/or bioactivates many endogenous compounds and is implicated in a range of cancers because of its ability to modify diverse promutagen and procarcinogen xenobiotics. The crystal structure of human SULT1A1 reported here is the first sulfotransferase structure complexed with a xenobiotic substrate. An unexpected finding is that the enzyme accommodates not one but two molecules of the xenobiotic model substrate p-nitrophenol in the active site. This result is supported by kinetic data for SULT1A1 that show substrate inhibition for this small xenobiotic. The extended active site of SULT1A1 is consistent with binding of diiodothyronine but cannot easily accommodate β-estradiol, although both are known substrates. This observation, together with evidence for a disorder-order transition in SULT1A1, suggests that the active site is flexible and can adapt its architecture to accept diverse hydrophobic substrates with varying sizes, shapes and flexibility. Thus the crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.

[6] Kinetics of slow and tight-binding inhibitors

Publisher Summary The interest in both slow and tight-binding inhibitors has been increasing, mainly, owing to their importance as chemotherapeutic agents, herbicides, and transition state analogs (reaction intermediate analogs). Although the quantitative description of the effects of these inhibitors poses special problems, it is worth emphasizing that there is nothing intrinsically different between the classical inhibitors and the tight-binding inhibitors. Usually, the derivation of the kinetic equations for these nonclassical inhibitors is carried out in the following way: first, an inhibition mechanism is proposed; second, some simplifying assumptions are set up; and, third, the corresponding kinetic equations are deduced. On the other hand, when the kinetic constants of a new (slow and/or tight-binding) inhibitor are to be determined, the following procedure is used: one or two alternative inhibition mechanisms are considered; the corresponding kinetic equations are fitted to the experimental data; and, finally, the model giving the best fit is accepted as correct and the corresponding kinetic constants taken as the real values. There are two extensive reviews about this type of inhibition, both of which include the kinetic aspects: the review by Williams and Morrison of tight-binding inhibition and the review by Morrison and Walsh about slow binding inhibitors. Thus, the present chapter discusses the aspects that have received less attention as well as some new developments, concerning the kinetics of slow and tight-binding inhibitors. Thus, it discusses a more general kinetic mechanism, the validity of commonly used simplifying assumptions, a procedure to determine the inhibition constants, and some more recent models for the analysis of slow and tight-binding inhibition.

Parameter estimation using a direct solution of the integrated Michaelis-Menten equation

A novel method of estimating enzyme kinetic parameters is presented using the Lambert omega function coupled with nonlinear regression. Explicit expressions for the substrate and product concentrations in the integrated Michaelis-Menten equation were obtained using the omega function which simplified kinetic parameter estimation as root-solving and numerical integration of the Michaelis-Menten equation were avoided. The omega function was highly accurate in describing the substrate and product concentrations in the integrated Michaelis-Menten equation with an accuracy of the order of 10(-16) when double precision arithmetic was used. Progress curve data from five different experimental systems were used to demonstrate the suitability of the omega function for kinetic parameter estimation. In all cases, the kinetic parameters obtained using the omega function were almost identical to those obtained using the conventional root-solving technique. The availability of highly efficient algorithms makes the computation of omega simpler than root-solving or numerical integration. The accuracy and simplicity of the omega function approach make it an attractive alternative for parameter estimation in enzyme kinetics.

The structure of human SULT1A1 crystallized with estradiol : An insight into active site plasticity and substrate inhibition with multi-ring substrates

Human SULT1A1 belongs to the supergene family of sulfotransferases (SULTs) involved in the sulfonation of xeno- and endobiotics. The enzyme is also one of the SULTs responsible for metabolic activation of mutagenic and carcinogenic compounds and therefore is implicated in various cancer forms. Further, it is not well understood how substrate inhibition takes place with rigid fused multiring substrates such as 17β-estradiol (E2) at high substrate concentrations when subcellular fractions or recombinant enzymes are used. To investigate how estradiol binds to SULT1A1, we co-crystallized SULT1A1 with sulfated estradiol and the cofactor product, PAP (3′-phosphoadenosine 5′-phosphate). The crystal structure of SULT1A1 that we present here has PAP and one molecule of E2 bound in a nonproductive mode in the active site. The structure reveals how the SULT1A1 binding site undergoes conformational changes to accept fused ring substrates such as steroids. In agreement with previous reports, the enzyme shows partial substrate inhibition at high concentrations of E2. A model to explain these kinetics is developed based on the formation of an enzyme·PAP·E2 dead-end complex during catalysis. This model provides a very good quantitative description of the rate versus the [E2] curve. This dead-end complex is proposed to be that described by the observed structure, where E2 is bound in a nonproductive mode.