Prospects & Overviews The biological significance of substrate inhibition: A mechanism with diverse functions 1) 2) 3) Michael C. Reed Ã, Anna Lieb and H. Frederik Nijhout Problems & Paradigms Many enzymes are inhibited by their own substrates, lead- The kinetics of an enzymatic reaction are typically studied by ing to velocity curves that rise to a maximum and then varying the concentration of substrate and plotting the rate of product formation as a function of substrate concentration. In descend as the substrate concentration increases. the conventional case this yields a typical hyperbolic Substrate inhibition is often regarded as a biochemical Michaelis-Menten curve, and a linear reciprocal Lineweaver- oddity and experimental annoyance. We show, using sev- Burk plot, from which the kinetic constants of the enzyme can eral case studies, that substrate inhibition often has be calculated. A surprisingly large number of enzymes do not important biological functions. In each case we discuss, behave in this conventional way. Instead, their velocity curves the biological significance is different. Substrate inhibition rise to a maximum and then decline as the substrate concen- tration goes up. This phenomenon is referred to as substrate of tyrosine hydroxylase results in a steady synthesis of inhibition, and it is estimated that it occurs in some 20% of dopamine despite large fluctuations in tyrosine due to enzymes [1]. A partial list of enzymes that show substrate meals. Substrate inhibition of acetylcholinesterase enhan- inhibition appears in Box 1. ces the neural signal and allows rapid signal termination. Substrate inhibition is often interpreted as an abnormality Substrate inhibition of phosphofructokinase ensures that that comes from using artificially high substrate concentration in a laboratory setting. In a review article on the mechanisms resources are not devoted to manufacturing ATP when it is of substrate inhibition in 1994, Kuehl [2] commented that plentiful. In folate metabolism, substrate inhibition main- ‘‘although recognized early on as an almost universal tains reactions rates in the face of substantial folate depri- phenomenon, it has nevertheless met an almost universal vation. Substrate inhibition of DNA methyltransferase disinterest. Probably the main reason for this neglect is that serves to faithfully copy DNA methylation patterns when the majority of enzymologists and many authorities in the field cells divide while preventing de novo methylation of regard substrate inhibition as being almost always a nonphy- siological phenomenon.’’ methyl-free promoter regions. There are several reasons for suspecting that substrate inhibition is not a pathological phenomenon, but a biologi- Keywords: cally relevant regulatory mechanism. First, in many cases .biological function; enzyme kinetics; substrate inhibition normal substrate concentrations are to the right of the velocity maximum, which indicates that these enzymes typically oper- ate under substrate inhibition. Second, many enzymes have specialized sites where a second substrate molecule can bind and act as an allosteric inhibitor. For those enzymes, substrate DOI 10.1002/bies.200900167 inhibition is clearly a specially evolved property. Third, evi- dence is accumulating that substrate inhibition plays critical 1) Department of Mathematics, Duke University, Durham, NC 27708, USA regulatory roles in a number of metabolic pathways. 2) Department of Mathematics, University of Colorado, Boulder, CO 80303, Substrate inhibition means that the velocity curve of a USA reaction rises to a maximum as substrate concentration 3) Department of Biology, Duke University, Durham, NC 27708, USA increases and then descends either to zero or to a non-zero asymptote. Many mechanisms are known that can result in *Corresponding author: Michael C. Reed such substrate-velocity curves [3, 4]. Here we discuss two E-mail: [email protected] simple mechanisms. Suppose that an enzyme, E, has two 422 www.bioessays-journal.com Bioessays 32: 422–429,ß 2010 WILEY Periodicals, Inc. ...Prospects & Overviews M. C. Reed et al. Box 1 A short list of enzymes that are subject to substrate Problems & Paradigms inhibition 4-hydoxyphenylpyruvate kynunrenine aminotransferase hydroxylase lactate dehydrogenase acetylcholinesterase L-amino acid oxidase adenosine 50-pyrophosphate lipoxygenase sulfurylase malate dehydrogenase adenosine kinase N-methyl transferase Figure 1. A reaction diagram for substrate inhibition. adenylate cyclase nucleotidediphosphate kinase aldehyde dehydrogenase O-acetylserine sulfhydrolase constant (1/K ) for the reaction S E S S E S. This alanine aminopeptidase octopine dehydrogenase N Á Á $ þ Á alcohol dehydrogenase PAPS synthetase is Haldane’s formula for substrate inhibition [5]. As there are aldehyde dehydrogenase phenol sulfotransferase two powers of [S] in the denominator, the velocity goes to zero aldose reductase prenyltransferase as [S] becomes large. Intuitively, this is because more and alkaline phosphatase purine nucleoside more of the enzyme is tied up in the unproductive ternary aminoacylase-I phosphorylase complex. aminoimidazolecarboximide pyrophosphatase ribotide pyruvate decarboxylase If we relax Haldane’s assumption and allow random-order transformylase pyruvate kinase binding of the substrates to the catalytically active and inac- arylamidase ribonuclease A tive sites, and allow 0 < k4 < k2, so the ternary complex can aspartate transcarbamylase ribonuclease T1 produce the product, but at a reduced rate, then one can carboxypeptidase ribonuclease T2 derive the velocity formula cholinesterase ribonucleoside diphosphate citrate synthase reductase S 2 cytochrome P450 (some) serine V k2 S k4 ½ ½ þ Ki 2 diamine oxidase hydroxymethyltransferase 2 E0 ¼ S S Km S ½ ½ diphospoglyceromutase sucrose-6-glycosyltransferase þ½ þk1Ki þ Ki DNA-methyltransferase sulfotransferases enolase trannsglucosyl-amylase In this case, the velocity curve rises to a maximum and esterase tRNA nucleotidyltransferase formyltetrahydrofolate trypsin then descends to E0k4 as [S] gets large. The value of Ki affects synthase tryptophan hydroxylase the shape of the velocity curves described by Equations (1) fructose-l,6-bisphosphatase tyrosine hydroxylase and (2). As Ki gets larger the peak moves to the right and the galactosyltransferase urease curve descends more slowly. Figure 2 shows the substrate gentamycin uridine kinase inhibition curves for tyrosine hydroxylase and tryptophan acetyltransferase xanthine oxidase hydroxylase. The Km values are the same but tryptophan glutamate dehydrogenase a-D-galactosidase hydroxylase has a much higher K value. As K , one glutathione reductase a-glucosidase i i !1 glycerol-3-phosphate b-fructofuranosidase regains the hyperbolic Michaelis-Menten curve. This makes dehydrogenase b-hydroxysteroid sense because when Ki is large there is very little enzyme tied HIV1-reverse transcriptase dehydrogenase up in the ternary complex. isocitrate dehydrogenase Our purpose here is not to describe the biochemical origins of substrate inhibition beyond this brief introduction. Rather, After Kaiser (1980) we want to discuss the biological functions of substrate inhi- bition. We use as our exemplars five enzymes that show substrate inhibition: tyrosine hydroxylase, acetylcholinester- binding sites for its substrate S, a catalytic site for binding that ase, phosphofructokinase, folate cycle enzymes, and DNA can produce the product, P, and a non-catalytic (or allosteric) methyltransferase. In each of these cases, substrate inhibition site that can produce the product at a reduced rate (see Fig. 1). plays a distinctly different regulatory role. The collection of We denote by E.S the substrate bound to the catalytic site, examples illustrates the broad diversity of physiological func- by S.E.S two substrate molecules bound to both the catalytic tions of substrate inhibition. and the non-catalytic site. Haldane [5] considered the simple case when a substrate molecule binds first to the catalytic site, followed by a substrate binding to the non-catalytic site, (as Tyrosine hydroxylase shown in Fig. 1), and assumed k4 0. Then, using the rapid equilibrium assumption, one can derive¼ the kinetic formula In the terminals of dopaminergic neurons in the central nerv- ous system, dopamine is synthesized in a two step process V k2 S from tyrosine L-Dopa dopamine. The enzyme that cata- ½ 2 1 ! ! E0 ¼ S lyzes the first step, tyrosine hydroxylase (TH), shows strong Km S ½ þ½ þ Ki substrate inhibition by tyrosine [6–8]. The velocity curve for where E0 is the total amount of enzyme present, Km the TH as a function of cytosolic tyrosine concentration is shown Michaelis constant, and Ki is the dissociation equilibrium in Fig. 2, and it has the typical substrate inhibition form. The Bioessays 32: 422–429,ß 2010 WILEY Periodicals, Inc. 423 M. C. Reed et al. Prospects & Overviews ... Figure 2. Substrate inhibition of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH). The velocity curve for TH as a function Problems & Paradigms of tyrosine concentration is shown using parameters (K 46, m ¼ Ki 160) obtained from fitting data [8]. The velocity curve for THP as¼ a function of tryptophan concentration uses parameters (K 46, m ¼ Ki 400) obtained by fitting experimental curves [12, 13]. For both tyrosine¼ and tryptophan, the range of normal daily average concen- tration is indicated on the X-axis. For TH the average concentration is near the point where the velocity curve has its maximum, but for