MODELLING OF METABOLIC PATHWAYS

Sym p. Biol. Hung. 18, pp. 123-142 (1974)

REGULATION OF KEY ENZYMES: STRATEGY IN REPROGRAMMING OF GENE EXPRESSION

GEORGE WEBER, NOEMI PRAJDA and JIM C. WILLIAMS

Laboratory for Experimental Oncology and Department of Pharmacology Indiana University School of Medicine, Indianapolis, Indiana, USA 46202

INTRODUCTION

The biochemical strategy of gene expression is manifested, in a large part at least, in the regulation of the activity, amount and isozyme pattern of key enzymes. The concept of regulation of the rate and direction of metab­

olism through control of key enzymes was developed in this Laboratory in studying hormonal regulation and the clinical and biochemical syndromes in metabolic diseases (Weber, 1959, 1963; Weber et al., 1965, 1966, 1971).

The experimental evidence obtained in this Laboratory supported the concept that the biochemical pattern in endocrine and nutritional regulation of metabolism could be understood in the control of opposing and competing key enzymes in antagonistic and competing synthetic and degradative pathways.

The principles learned in these investigations were also applied to studies on the sequential unfolding of the pattern of gene expression that occurs in regeneration and in neoplasia (for review see Weber, 1974).

Previous studies pointed out the operational advantage of this concept that proposed a pattern of behavior and control mechanisms for the various key enzymes, the operation of which was readily subject to experimental testing. Thus, this concept provided a set of predictions for the antici­

pated antagonistic behavioral pattern of key enzymes in physiological and pathological conditions, such as in hormonal regulation (steroid and i n ­ sulin action, in diabetes, etc.), in metabolic diseases (Glycogen Storage Disease), in differentiation and in neoplasia. A useful insight yielded by this approach proved to be the experience that from determining a single pair of such antagonistic enzymes, e.g., phosphofructokinase/fructose-1,6- diphosphatase, one was able to predict the behavior of other antagonistic pairs of key enzymes in carbohydrate metabolism (glucose-6-phosphatase/

glucokinase, pyruvate carboxylase/pyruvate kinase) under various conditions entailing alterations in homeostatic balance. The experimental and con­

ceptual studies led to the formulation of the general theory of the molec­

ular correlation concept which was first tested in examining the behavior of carbohydrate metabolism under various conditions involving reprogramming of gene expression (Weber, 1973). The special theory of the molecular correlation concept refers to studies on the alterations of gene expression in neoplasia (Weber, 1974). Detailed investigations carried out in this Laboratory demonstrated that the regulation of the rate and direction of opposing pathways of synthesis and degradation through control of antagon­

istic key enzymes was applicable not only to carbohydrate but also to

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Table 1

Pleiotropic action of insulin on hepatic enzymes of different metabolic pathways

FUNCTIONS INCREASED ENZYMES INDUCED

GtycogeneA-ib Glycogen synthetase

GlyeotyiiÁ Glucokinase (high К isozyme)

Phosphofructokinase

Pyruvate kinase (high isozyme)

Lipogeneiti Citrate cleavage enzyme

Acetyl CoA carboxylase Fatty acid synthetase

SIAOPH production G lucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Malate enzyme

Pent оде. phosphate patmiay Transaldolase Transketolase Thymidine. incoaporation into ONA DNA polymerase

Thymidine degradation to CO^ Thymidine phosphorylase

FUNCTIONS PECREASEP ENZYMES SUPPRESSED

GLuconeogenebit Glucose-6-phosphatase

Fructose-1,6-diphosphatase Phosphoenolpyruvate carboxykinasé Pyruvate carboxylase

Urea cycle Ornithine carbamyltransferase

Arginine synthetase Argininosuccinase

G L Y C O L Y S I S K R E B S C Y C L E I--- 1 I--- --- 1

Fig. 1. Insulin: integrative action at the hepatic enzyme level.

GK=Glucokinase, PFK=Phosphofructokinase, PK=Pyruvate kinase, HK=Hexokinase, G6P DH=Glucose-6-phosphate dehydrogenase, 6-PG DH=6-Phosphogluconate dehydrogenase, IDH=Isocitrate dehydrogenase, F-ASE=Fumarase, ME=Malic enzyme, AC=Acetyl CoA carboxylase, CS=Citrate synthase, TA=Transaldolase,

TK=Transketolase.

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pyrimidine, DNA, ornithine, and membrane cAMP metabolism (Weber et al., 1971, 1972, 1973, 1974; Ferdinandus et al., 1971; Williams-Ashman et al., 1972) .

The purpose of this presentation is to exairçine in more detail the key enzyme concept, the selective advantages and economy the key enzymes p r o ­ vide for the organism and the applicability of these principles to purine metabolism and the utilization of UDP.

MATERIALS AND METHODS

The conceptual significance of the role of key enzymes in the regula­

tion of metabolism and in the control of gene expression was outlined else­

where (Weber, 1973). The description of the strains and conditions of the expression and evaluation of the enzymatic data are provided elsewhere

(Weber, 1974).

RESULTS AND DISCUSSION

Pleiotropic Action of Insulin on Hepatic Key Enzymes

It was discovered earlier in this Laboratory that the integrative action of insulin at the molecular level in the liver entailed an antagon­

istic action on the biosynthesis of groups of key enzymes opposing each other in gluconeogenesis and in glycolysis. Subsequent work pointed out that the hepatic action of insulin involved reprogramming of gene expres­

sion and evidence was provided that this process included a shift in the isozyme pattern of the key glycolytic enzymes (for review see Weber, 1975) . Recently, we obtained extensive evidence by measuring the activity and the immunoprecipitable isozyme concentration of PFK that the concentration of this enzyme was decreased in low insulin states (starvation, diabetes) and high insulin states (refeeding, insulin administration) restored the con­

centration of PFK to normal level (Dunaway § Weber, 1974, 1974a; Weber, 1975a). The determination of PFK concentration by two independent methods strongly supported the conceptual view we proposed for the integrative action of insulin at the molecular level. Further investigations in this Laboratory and in other Centers revealed that there are also characteristic alterations in enzyme activity of the pentose phosphate pathway, glyco- genesis, thymidine metabolism and the urea cycle that occur in diabetes and insulin returns the activities to normal range. Table 1 and Figure 1 sum­

marize the array of enzymes induced and those suppressed by insulin action.

It was proposed elsewhere that the reprogramming of gene expression occurring as a result of insulin administration that restores enzyme activ­

ities to normal range might operate through determining the expression of master genes capable of exerting pleiotropic action on groups of function­

ally related key enzymes operating in different metabolic pathways (Weber et al., 1974). There are other levels of controls where pleiotropic regu­

lation might be achieved and these alternative possibilities were discussed in a recent paper by Weber et al. (1974). The term pleiotropy, as it re­

fers to reprogramming in gene expression through hormonal influences or in

neoplasia, was also discussed in the same article. The importance of the multienzyme alterations is relevant for the present discussion which deals with the biochemical strategy of the cell as it is expressed through regu­

lation of key enzymes.

Through the operation of hormonal influences, adaptation in a mammalian system has reached the highly advanced integrated state that provides sel­

ective advantages for the system. When experimentally or clinically endo­

crine alterations occur such as in diabetes, marked quantitative and quali­

tative shifts take place in the ratios of key enzymes in different path­

ways. Administration of the hormone that was in short supply, e.g., insul­

in, is capable of restoring the homeostatic balance to normal range. Thus, the reprogramming of gene expression in endocrine alterations has a rever­

sible nature and the alterations themselves appear to be not heritable.

The situation is entirely different in neoplasia where the alterations in gene expression that occur appear to be irreversible and heritable. In the following we will examine the behavior of key enzymes in two areas that we have been studying recently: purine and pyrimidine metabolism.

Regulation of Purine Metabolism: Control of Glutamine PRPP Amidotransfer­

ase Activity

The opposing pathways of synthesis and degradation of IMP are shown in Figure 2. In this Figure we emphasize that the origin of the de novo syn­

thesis of IMP starts at the product of the pentose phosphate pathways, ribose-5-phosphate. Previous work from this Laboratory emphasized this strategic link between carbohydrate and purine metabolism accomplished by PRPP synthetase that converts ribose-5-phosphate into PRPP (Weber et al., 1974) . These studies resulted in the discovery that PRPP synthetase was increased in the rapidly growing tumors (Heinrich et al., 1974); thus, the reaction involved in the de novo synthesis of PRPP was increased, leading to a heightened potential for IMP production. With the discovery of the increased PRPP synthetase activity it became of immediate interest to el­

ucidate the behavior of the enzyme that utilizes PRPP into the de novo purine biosynthesis: glutamine PRPP amidotransferase, amidophosphoribosyl- transferase, EC 2.4.2.14, (amidotransferase) .

The comparison of the kinetic behavior of the liver and hepatoma amidotransferase and the development of an assay system applicable tq kin­

etic conditions of rat liver and hepatomas was published recently from this Laboratory (Katunuma and Weber, 1974; Weber et al., 1975). An anal­

ysis of the molecular properties of amidotransferase from normal rat liver and fron rapidly growing hepatomas is of interest in evaluating the role of this key enzyme in metabolic regulation.

Figure 3 indicates that the affinity of the normal liver amidotrans­

ferase to PRPP yields a sigmoid curve, whereas the hepatoma enzyme exhib­

its -Michaelis-Menten kinetics. These results indicate that the liver en­

zyme had very low activity at a PRPP concentration of less than 1 mM, but in the same substrate range the hepatoma amidotransferase activity was high. Thus, the tumor enzyme was more readily saturated by PRPP at the

low level that may occur in the tissues (Weber et al., 1975; Prajda et al., 1975) .

The biological significance to the neoplastic transformation is also determined by the responsiveness of the tumor enzyme to physiological reg­

ulatory signals. Earlier work by Katunuma and Weber showed that the am­

idotransferase in the rapidly growing hepatoma was much less sensitive to the action of the physiological inhibitor, AMP (1974).

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Inosinicase

atomas. PRPP synthetase was increased in rapidly growing hepatomas and amidotransferase was elevated in all hepatomas; the catabolic enzymes, 5'-nucleotidase and xanthine oxidase, were decreased in rapidly growing hepatomas. The resulting metabolic imbalance fav­

ors purine biosynthesis and should prevent recycling of metabol­

In document REGULATION OF METABOLIC MODELS MATHEMATICAL 18 (Pldal 123-130)