Metabolic Control Analysis from a Control Theoretic Perspective

Brian P. Ingalls Department of Applied Mathematics University of Waterloo Waterloo, Ontario Canada N2L 3G1
bingalls@math.uwaterloo.ca

Abstract A theory of control of biochemical systems developed by the theoretical biology community is interpreted from a control-theoretic point of view. This theory, known as Metabolic Control Analysis (MCA), is based on an extension of parametric sensitivity analysis to stoichiometric systems. The main results of MCA are shown to correspond to particular solutions of an appropriately stated tracking problem. This presentation leads to some natural generalizations of existing results.

I. I NTRODUCTION Issues of regulation and control are central to the study of biochemical systems. The maintenance of cellular behaviour and the appropriate response to environmental signals can only be achieved by systems which have evolved to be robust against certain perturbations and sensitive to others. These behaviours demand the use of feedback. It is then natural to expect that the tools of feedback control theory, which were developed to address the analysis and design of self-regulating systems, would be useful in the study of these biological networks. Biochemical mechanisms for implementation of feedback control were rst discovered in the biosynthetic pathways of metabolism, and it was within the study of metabolism that a quantitative theory of control and regulation of biochemical networks was rst developed. The fundamental tool used in this study is parametric sensitivity analysis, applied primarily at steady state. One approach, dubbed Metabolic Control Analysis (MCA), or sometimes Metabolic Control Theory (MCT), makes use of a standard linearization technique in addressing steady state behaviour [5], [6], [11]. This paper will focus on the straightforward linearization method used by the MCA community. This framework provides a direct connection between these biochemical studies and the general theory of parametric sensitivity analysis. Moreover, linearization leaves intact the stoichiometric relationships which are exploited in studies of these chemical networks. Indeed,

as will be shown below, it is this stoichiometric nature which distinguishes the mathematics of MCA from standard sensitivity analysis. As rst shown in [17], application of some basic linear algebra provides an extension of sensitivity analysis which captures the features of stoichiometry. Beyond these mathematical underpinnings, the eld of MCA deals with myriad intricacies of application to biochemical networks that demand careful interpretation of experimental and theoretical results. Surveys can be found in [3], [4], [7]. The analysis in this paper is based on the standard ordinary differential equation-based description of biochemical systems in which the states are the concentrations of the chemical species involved in the network and the inputs are parameters inuencing the reaction rates. In most cases this model is a nonminimal representation of the system. In addressing metabolic systems, it is standard to take enzyme activity as the parameter input. This choice of input typically results in an overactuated system with more inputs than states. However, in addition to the species concentrations, there are outputs of primary importance, namely the rates of the reactions within the network. As will be described below, it is a consequence of the stoichiometric nature of the system that the degree of overactuation coincides exactly with the degree by which this reaction rate output is underdetermined as a function of the state. The result is a control system in which the output enjoys some autonomy from the state. Consequently, issues of controlling the state and output can be, to a degree, addressed separately. Many investigations of metabolic redesign have appeared in the MCA literature (e.g. [12], [19], [21]). These results can all be seen as contained within
Enzymes are proteins which act as catalysts. In most biochemical networks, each reaction is catalysed by a single enzyme and the reaction rate is directly proportional to the level of enzyme activity.

the metabolic design approach developed by Kholodenko et al. [13], [14]. The current paper describes these results as solutions to a tracking problem, thus drawing parallels between MCA and control theory as well as providing control-theoretic generalizations of the main results of MCA, the Summation and Connectivity Theorems. II. P RELIMINARIES Consider n chemical species involved in m reactions in a xed volume. The concentrations of the species make up the n-dimensional vector s. The rates of the reactions are the elements of the m-vector v. These rates depend on the species concentrations and on a number of parameters which are collected in a k -vector p. The function v = v(s, p) is assumed continuously differentiable. The network topology is described by the n m stoichiometry matrix N, whose i, j -th element indicates the net number of molecules of species i produced in reaction j (negative values indicate consumption). The system dynamics are described by d s(t) = Nv(s(t), p(t)) for all t 0. (1) dt In addition to the state, s(), and the input p(), the variables of primary interest in this system are the reaction rates v(s, p). Thus, in interpreting (1) as a control system, we choose an output y comprised of these two vectors: y(s, p) = s v(s, p) . (2)

of such a system it would be worthwhile to begin with a reduction afforded by linear dependence. A. Deciencies in row rank As mentioned, structural conservations in the reaction network reveal themselves as linear dependencies among the rows of the stoichiometry matrix N. Let r denote the rank of N. Following the standard treatment (presented in [17]), relabel the species so that the rst r rows of N are independent. The species concentration vector can then be partitioned T T r as s = (sT i , sd ) , where si R is the vector of independent species and sd Rnr contains the dependent species. Next N is partitioned into two submatrices. Calling the rst r rows NR , we can write N = LNR where the matrix L is referred to as the link matrix. Following [17] we arrive at a reduced version of (1) d si (t) = NR v(Lsi (t) + T, p(t)) (3) dt where T depends on the initial condition, and s(t) = Lsi (t) + T. It follows that the n-dimensional state enjoys only r degrees of freedom. Thus the original description in terms of n state variables is nonminimal (provided r < n), regardless of the form of the reaction rates. B. Deciencies in column rank Recalling that r denotes the rank of the stoichiometry matrix N, relabel the reactions so that the rst m r columns of N are linearly dependent on the remaining r. Partition the vector of reaction rates v correspondingly into m r independent (vi ) and r T T T dependent (vd ) rates as v = (vi , vd ) . In analogy to the procedure outlined above, one might hope to reach a reduced description of the system dynamics in which some of these reaction rates are eliminated, but this is an impossible task. (Such an elimination could decouple an input channel from the dynamics.) However, an analogous reduction can be made after a change of variables in the reaction rate space Rm . To generate such a transformation, begin by choosing a matrix K of the form K= Imr K0

Linearly dependent rows within the stoichiometry matrix correspond to integrals of motion of the system. Each redundant row identies a chemical species whose dynamics is completely determined by the behaviour of other species in the system through a conservation relation. The consequences of linear dependence among the columns of N will be explored below. If the stoichiometry matrix has full column rank then steady state can only be attained when v(s, p) = 0. Most biochemical systems admit steady states in which there is a ux through the network. These correspond to non-zero reaction rate vectors v which lie in the nullspace of N. III. C ONSEQUENCES OF R ANK D EFICIENCIES Networks which describe metabolic systems often have highly redundant stoichiometries. As an example, consider a metabolic map from Escherichia coli published in [18] which has a 770 931 stoichiometry matrix of rank 733. Clearly, in attempting an analysis

such that the columns of K form a basis for the kernel of N. Next, introduce the invertible m m matrix P= Imr K0 0(mr)r Ir ,

and dene the transformed reaction rates (s, p) by (s, p) = P1 v(s, p).

It will prove useful to partition these transformed rates into independent (i Rmr ) and dependent (d T T T ) . Rr ) components: = (i , d C Letting N denote the submatrix of N consisting of the last r columns, the dynamics (1) can be written in terms of the transformed rates as d s(t) = NP (s(t), p(t)) dt = [0n(mr) NC ] (s(t), p(t)) = NC d (s(t), p(t)). (4) We conclude that the values of the independent transformed rates have no bearing on the system dynamics. To relate this fact to the true reaction rates v, note that i = vi . Thus this transformation characterizes the r combinations of reaction rates which impact the dynamics of the state (i.e. d = vd K0 vi ) while identifying m r rates whose values can be set independently of the dynamics (i.e. i = vi ). This explicit transformation in reaction rates is a novel construction. However, the decomposition of the reaction rate vector into independent and dependent parts is standard, as outlined in, e.g., [7]. In most work on MCA, attention is restricted to the values of the reaction rates at steady state, which are referred to as the system uxes J. By partitioning N as described above, the vector J is separated into dependent and inT T dependent components J = (JT i , Jd ) corresponding to the partitioning of the reaction rates. These steady state rates satisfy J = KJi = Imr K0 Ji .

Complete authority over the evolution of the independent species concentrations lies with the dependent transformed rates d = vd K0 vi . To force the output to track the target according to (vi (t), si (t)) (r1 (t), r2 (t)) the input p() must satisfy vi (Lr2 (t) + T, p(t)) = r1 (t), vd (Lr2 (t) + T, p(t)) = K0 r1 (t) +
1 NC R

This is an immediate consequence of (4). C. Input-Output Interpretation System (1) enjoys only m degrees of freedom in the m + n-dimensional output (2). This could be described directly by observing that the m reaction rates are free to vary independently, while any such choice prescribes the evolution of the n species concentrations. The proceeding analysis has described a more useful characterization of freedom in r independent concentrations and m r independent reaction rates. We now address how the motion in these m degrees of freedom might be inuenced by the parameter input p(). It will be illustrative to consider an overly ambitious control objective. Suppose given an mdimensional signal r(), partitioned into r1 () Rmr and r2 () Rr , dened for t 0. Consider the problem of forcing the system to track r perfectly, in the sense that vi (t) r1 (t) and si (t) r2 (t). Clearly this will only be possible if r() satises certain conditions.

for all t 0. Equations (5) and (6) represent an extreme control objective, but serve to illustrate the possibility of addressing the behaviour of the independent concentrations and reaction states rates separately. A solution could be reached by solving these m equations in p at each time t. In the absence of redundancy this is infeasible if the vector p has fewer than m components. In the metabolic setting, the standard choice of enzyme activities as parameter inputs typically yields precisely m input channels. Since each enzyme (typically) catalyses a single reaction, this results in a set of reaction-specic parameters, i.e. each reaction rate vj (s, p) depends on exactly one parameter: vj = vj (s, pj ). In this case the system of equations in (5) and (6) takes on a simple uncoupled form. Analysis of the complete authority control objective of (5) and (6) is a purely academic exercise our ability to manipulate biochemical systems falls far short of the requirements determined above. We next consider a special case of this open-loop tracking problem which is of more signicance in a metabolic setting. Keeping in mind the application of metabolic redesign, consider the problem of asymptotic tracking of a constant target r = (r1 , r2 ) Rm . We will neglect issues of stability and address the problem of choosing a constant parameter input which introduces a steady state satisfying vi = r1 , si = r2 . Specializing the proceeding analysis to this case, we see that the parameter vector p must satisfy v(Lr2 + T, p) = Kr1 . (7) This steady-state tracking problem has been addressed by Kholodenko et al. in [13], [14]. Under the assumption that the parameters are reaction-specic and appear multiplicatively, the solution is immediate, as pointed out in [13]. A discussion of biochemically relevant cases where those assumptions are relaxed can be found in [14]. While the system of m equations in (7) cannot be treated in general, a local description of the solution can be investigated regardless of the form of the reaction rates, as follows. Let p0 be a nominal parameter value corresponding to a steady state species

(5) d r2 (t) (6) dt

v 0 0 concentration s0 . Provided the matrix p |(s ,p ) is invertible, equation (7) can be used to locally dene the parameter input as a function of r, i.e. p = p(r). In some cases it may be possible to solve for the function p(r). More generally, the function can be characterized locally by differentiating (7) at (s0 , p0 ), which gives

time-varying behaviour. In particular, at steady state, an assumption of asymptotic stability is not restrictive, since unstable steady states are not observed in the lab. At steady state, the (reduced) system dynamics give 0 = NR v(Lsi + T, p).
0

(9)

v p v v p = K and L+ = 0. p r1 s p r2 In terms of the vector r = (r1 , r2 ), this can be written as v p v = [K L]. (8) p r s The condition can be put in a more standard linear setting by redening r to describe the displacement of the set point from the nominal position. Equation (8) then gives a local description of the parameter p(r) required to track the point (vi (s0 , p0 ), s0 i ) + r. When interpreted in this manner, (8) provides a link between the results in this section and the main results of MCA. IV. M ETABOLIC C ONTROL A NALYSIS The eld of Metabolic Control Analysis was born in the mid-70s out of the work of Kacser and Burns [11] and Heinrich and Rapoport [5], [6]. These two groups independently arrived at an analytical framework for addressing questions of control and regulation of metabolic networks. Specically, these papers outlined a parametric sensitivity analysis around a steady state and also presented relationships between the sensitivity coefcients. These relations, known as the Summation and Connectivity Theorems, have been used to provide valuable insights into the behaviour of metabolic pathways. Since its inception MCA has been used successfully in the study of a great many metabolic systems. In addition to elucidating these biochemical mechanisms, this sensitivity analysis allows prediction of the effects of intervention. As such, it is a powerful design tool which has been adopted by the metabolic engineering community (cf. e.g. [2], [15], [20]) and has been used in rational drug design (cf. e.g. [1], [2]). We follow the formalism developed in [17] (see also [7], [9]). Beginning with system (1), follow the decomposition described in Section III-A to arrive at a row reduced system of the form (3). As mentioned above, MCA is primarily concerned with local behaviour around an asymptotically stable steady state. While this is the simplest behaviour to address analytically, the primary motivation for this narrow range of focus is the difculty of experimentally analysing

Suppose given a nominal parameter value p and a corresponding steady state s0 . Under the assumption of asymptotic stability, (9) yields a local implicit description of si as a function of p, with si (p0 ) = s0 i. To determine the effect of small parameter changes on this steady state si (p), differentiate (9) with respect to p at the nominal point to yield 0 = NR v v dsi L + s dp p .

The sensitivities in species concentration, referred to as unscaled independent concentration response coefcients are the components of the matrix si := d si (p) = NR v L R p dp s
1

NR

v . (10) p

This calculation can be extended to the complete si . Per s := LR concentration vector s by dening R p p turbations in the initial conditions can be treated along similar lines (see, e.g. [9]) but will not be addressed here. The use of the specialized terminology response for the coefcients in (10) was introduced to distinguish these system sensitivities (total derivatives) from component sensitivities (partial derivatives) which will be introduced below as elasticities. In addition to this sensitivity in the state variables, the sensitivities of the reaction rates, referred to as the unscaled ux response coefcients, are also of interest: v R p := = d v(s(p), p) dp v v L NR L s s

NR

v v + . p p

The rst m r rows of this matrix constitute the in vi := dependent unscaled ux response coefcients R p d dp vi (s(p), p). These response coefcients represent absolute sensitivities. In application it is the relative sensitivities, reached through scaling by the values of the related variables, that provide useful measures of system behaviour. In the biochemical literature (and especially in addressing experimental data) the scaled versions are preferred. On the other hand, from a mathematical point of view, the unscaled sensitivities can be seen as more fundamental. For that reason, we will deal with unscaled coefcients in what follows. The response coefcients describe the asymptotic response of the linearized system to (step) changes in

the parameter vector p. As such, they can be used to predict the asymptotic effect of small changes in the parameter values. However, in using sensitivity analysis to address the inherent behaviour of a network, it is often more useful to ignore the details of the actuation and identify the reaction rates directly with the parameters. In addressing absolute (unscaled) sensitivities, this amounts to supposing that the reaction rates depend on the parameters specically and v directly: p = I. Under this assumption the responses dened above are referred to as the unscaled control coefcients of the system. These are the primary objects of interest in MCA as they provide a means to quantify the dependence of system behaviour on the individual reactions in the network. They are dened by s C v C := := L NR v L s
1

In terms of the independent and dependent variables, this statement can be written as vi C 1 s L . si = K C B. The Theorems of MCA: an Input-Output Interpretation Several researchers have investigated the consequences of the Theorems for metabolic redesign. The Universal Method of Kacser and Acerenza [12] describes coordinated parameter changes which lead to increased ux without affecting metabolite concentrations. The complementary problem of altering species concentrations without affecting steady state uxes was addressed in [19] using the Connectivity Theorem. A local treatment of the general output tracking problem was presented in [21] and will be highlighted below. To determine the consequences for system behaviour of the Theorems (13), (14), one can observe that these equations describe response coefcients corresponding to parameter perturbations of a particular form. Such statements are not directly useful in addressing issues of controlling system (1) since the parameters (i.e. the input channels) are specied directly in the model. It will be helpful introduce a new input variable and generalize the notion of response coefcient to allow description of the effect of simultaneous changes in multiple parameters. To that end we introduce a q -dimensional input space and call a function p : Rq Rm a coordinated parameter input. The denition of system response can be extended to inputs u Rq as the sensitivity of the system to perturbations of the form p = p(u): d s(p(u)) du v := d v(s(p(u)), p(u)) R u du When q = m and p() is the identity map, these reduce to the responses dened earlier. When stated in terms of coordinated parameter inputs, the Theorems of MCA can be given direct interpretations as tools for network design since they specify which choice of coordinated parameter input p(u) will produce a desired effect. Summation Theorem: Suppose a coordinated paramv eter input p(u) is chosen such that u lies in the v nullspace of N, i.e. u = KA1 for some (m r) q matrix A1 . Then s R u := si = 0, R u vi = A 1 . R u Connectivity Theorem: Suppose a coordinated pav rameter input p(u) is chosen such that u lies in the

NR
1

(11)

v v L NR L NR + Im . (12) s s To make the distinction between component and system sensitivities explicit, the partial derivatives of v are referred to as the elasticities of the system. Specically, dene the unscaled substrate elasticity v v p := s := s and the parameter elasticity p. A. Sensitivity Invariants: The Theorems of MCA The stoichiometric nature of a biochemical network imposes invariants on the sensitivity coefcients. Descriptions of these invariants originally appeared in the papers by Kacser and Burns [11] and Heinrich and Rapoport [5], [6] and have since been generalized and extended. These results, known as the Summation Theorem and the Connectivity Theorem will be stated next. The Summation Theorem was stated in general form by Reder in [17] in which it is observed that if the matrix K is chosen as in Section III so that the columns of K lie in the nullspace of N, then sK = 0 v K = K, C C (13) which follows directly from the denitions of the control coefcients (11), (12). Reder [17] generalized the original form of the Connectivity Theorem to the algebraic statements s v C s L = L C s L = 0, (14) which again follow directly from (11), (12). These results can be stated simultaneously in the Control Matrix Equation as given in [8]: combining (13) and (14) gives v K 0 C K s L = . s 0 L C

column space of s L, i.e. r q matrix A2 . Then si R u = A2 ,

v u

= s LA2 for some = 0.

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vi R u

Together, these statements provide an alternative version of the control matrix equation. Control Matrix Equation: The response to any coordinated parameter input p(u) can be characterized as follows: Since the columns of [K s L] form a basis for Rm , there exists a unique m q matrix v T T A = [AT s L]A, and 1 , A2 ] such that u = [K
si i Rv u = A1 , Ru = A2 .

(15)

A geometric interpretation of this result was presented in [16]. In this design form, the Theorems provide a transparent solution to the local tracking problem. To make an explicit connection to Section III, consider a coordinated parameter input p() which is linear and satises p(0) = p0 . Then, if the parametric sensitivities satisfy (15), a constant input u in the linearization of (1) leads to a steady state (vi , si ) = (vi (s0 , p0 ), s0 i )+ Au. It was determined in Section III that to track a steady state satisfying (vi , si ) = (vi (s0 , p0 ), s0 i ) + r we must take p = p(r) satisfying v p = [K s L]. p r (16)

This condition coincides with (15) when q = m, A = Im and u = r. (An alternative derivation of these sensitivity results can be reached by addressing the linearization of system (1) directly as carried out in [10].) We conclude that the discussion in Section III can be seen as providing a generalization of the Theorems of MCA to tracking of time-varying targets in the original nonlinear system. V. C ONCLUSION The discussion in this paper provides a controltheoretic interpretation of the mathematical basis of MCA, but addresses only a small part of the work in that area. Once the mathematical preliminaries are in place, the bulk of the analysis comes in application: developing useful models, identifying important questions, and interpreting the results. Acknowledgments The author would like to thank Herbert Sauro, Christopher Rao, and Matthew Scott for helpful discussions. This work was funded by a Discovery Grant from the Canadian Natural Sciences and Engineering Research Council.