98

cholesterol and cholesteryl ester by hepato- cytes and generation of H D ~ particles a3. In support of these findings there is some evi- dence that the liver preferentially uses and excretes the cholesterol of HDL zT. There is also evidence that HDL delivers cholesterol to the adrenals and gonads, the other organs in man that are capable of metabolizing cholesterol 6, 29.

Acknowledgements

We thank the Wellcome Trust and the British Heart Foundation for financial sup- port for much of our work and Dr G. Schmitz and Dr J. A. Glomset for fruitful discussions on aspects of HDL metabolism.

References 1 Jackson, R.L., Morrisett, J.D. and Gotto,

A. M. Jr. (1976)PhysiolRev. 56, 259-316 2 Katagiri, C., Owen, J.S., Quinn, P.J. and

Chapman, D. (1981) Eur. J. Biochem. 118, 335-338

3 Owen, J. S. Nature (London), 292, 106 4 Stein, Y. and Stein, O. (1980) inAtherosclerosis

V (Gotto, A. M. Jr., Smith, L. C. and Allen, B., eds), pp. 653-665, Springer-Verlag, New York

5 Albers, J. J., Cheung, M. C., Ewens, S. L. and Tollefson, J. H. (1981)Atherosclerosis 39, 395-409

6 Brown, M. S., Kovanen, P. T. and Goldstein, J. L. (1981)Science 212, 628-635

7 Hui, D. Y., Innerarity, T. L. and Mahley, R. W. (1981)J. Biol. Chem. 256, 5646-5655

8 Malloy, M. J., Kane, J. P., Hardman, D. A., Hamilton, R. L. and Dalai, K. B. (1981)J. Clin. Invest. 67, 1441-1450

9 Quarfordt, S. H., Shelborne, F. A., Meyers, W., Jakoi, L. and Hanks, J. (1981) Gastroenterology, 80, 149-153

10 Kane, J. P., Hardman, D. A. and Paulus, H. E. (1980) Proc. Natl Acad. Sci. U.S.A. 77, 2465-2469

11 Myant, N. B. (1981 ) The Biology of Cholesterol and Related Sterols , Heinemann, London

12 Brown, M. S. and Goldstein, J. L. (1979) Proc. Natl Acad. Sci. U.S.A. 76, 3330-3337

13 Schonfeld, G., Patsch, W., Pfleger, B., Witztum, J. L. and Weidman, S. W. (1979)J. Clin. Invest. 64, 1288-1297

14 Florin, C.-H., Albers, J. J., Kudchodkar, B. J. "and Bierman, E. L. (1981)J. Biol. Chem. 256, 425-433

t5 Weisgraber, K. H. and Mahley, R. W. (1980) Z LipidRes. 21,316-325

16 Pittman, R. C., Attie, A. D., Carew, T. E. and Steinberg, D. (1979) Proc. Natl Acad. Sci. U.S.A. 76, 5345-5349

17 Schwartz, C.C., Halloran, L.G., Vlahcevic, Z. R., Gregory, D.H. and Swell, L. (1978) Science 200, 62-64

18 Tall, A. R. and Small, D. M. (1979)Adv. Lipid Res. 17, 1-51

19 Nicoll, A., Miller, N. E. and Lewis, B. (1979) Adv. Lipid Res. 17, 53-106

20 Daniels, R. J., Guertler, L. S., Parker, T. S. and Steinberg, D. (1981) J. Biol. Chem. 256, 4978--4983

21 Anderson, D. W., Nichols, A. V.,Pan, S. S. and Lindgren, F. T. (1978)Artherosclerosis 29, 161-179

22 Glomset, J. A., Mitchell, C. D., King, W. C.,

Appelgate, K. A., Forte, T., Norum, K. R. and Gjone, E. (1980) Ann. N.Y. Acad. Sci. 348, 224-243

23 Eisenberg, S. (1980)Ann. N.Y. Acad. Sci. 348, 30--47

24 Glomset, J. A. (1978) inHigh Density Lipopro- teins and Atherosclerosis (Gotto, A.M. Jr., Miller, N. E. and Oliver, M. F., eds), pp. 57--65, Elsevier/North-Holland Biomedical Press, Amsterdam

25 Fielding, C. J. and Fielding, P. E. (1981)J. Biol.

T1BS - March 1 982

Chem. 256, 2102-2104 26 Marcel, Y. L., Vezina, C., Emond, D. and Suzue,

G. (1980)Proc. Natl Acad. Sci. U.S.A. 77, 2969-2973

27 Schmitz, G., Assmann, G. and Melnik, B. Clin. Chim. Acta (in press)

28 Van't Hooft, F. M., Van Gent, T. and Van Tol, A. (1981)Biochem. J. 196, 877485

29 Brown, M. S., Kovanen, P. T. and Goldstein, J.L. (1979) Rec. Prog. Hormone Res. 35, 215-257

Control of the circadian rhythm in pineal serotonin N-acetyltransferase

activity: possible role of protein thiol :disulfide exchange David C. Klein and M. A. A. Namboodiri

The activity o f p ineal serotonin N-acetyltransferase, which is under neural control, is 30- to 70-fold higher at night than dur ing the day. Exposure to light at night causes a rapid (tt = 3--4 rain) decrease in activity. There is reason to suspect this decrease m a y

result f r o m a protein th ioh disulf ide exchange.

Pineal serotonin N-acetyltransferase plays a role in the conversion of tryptophan to melatonin, an apparent antigonadotropic hormone of the pineal gland (Fig. 1) ~,z. Interest in this enzyme started to grow when it became clear that it controls large changes in melatohin concentration during the course of a day (Fig. 2) 3. Interest inten- sifted as it became apparent that a complex neural-biochemical system exists to regu- late specifically the activity of this enzyme 2,4,3. Many aspects of this system have been described 1,~, and current atten- tion is focused on the molecular mechan- isms which control the enzyme. This article will review the neural-biochemical regula- tion of N-acetyltransferase, and our prop- osal that the extraordinarily rapid (tt = 3---4 win.) physiological inactivation of this enzyme 3 is the result of a protein thiol: dis- ulfide exchange.

The neural control system The neural signals which control the

daily changes in N-acetyltransferase activ- ity appear to originate in the suprachiasma- tic nucleus (SCN) of the hypothalamus (Fig. 3; Ref. 5). This structure functions as a 24 h biological clock and stimulates the pineal gland for a 10 h period at night. The 24 h cycle in the SCN is kept entrained with

David C. Klein and M. A. A. Namboodiri are at the Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205, U.S.A.

the environmental lighting cycle by a direct neural connection with the eye. It seems as

~ CHICH(NH2)COOH

H TRYPTOPHAN

~ Tryplophan hydroxylase

H O ~ - - ~ CH2CH(NHzlCOOH

H

5 HYDROXYTRYPTOPHAN

I Aromatic amino acpd decafboxylase

H O ~ - - - ~ CHzCHzNH 2

H SEROTONIN

(5 Hydroxylryptamine) I N-AcetyItransferase

H O ~ CHzCH2NHCOCH 3

H N ACETYLSEROTONIN

(5 Hydroxy N acetyqtryptarninet I Hydroxyindole-O-methyltransferase

C H ~ O ~ CH2CHzNHCOCH3

H

MELATONIN (5 Methoxy N acetyltryptaminel

Fig. 1. The enzymatic conversion of tryptophan to melatonin.

T I B S - March 1982

if the SCN clock is reset daily and without this daily re-adjustment, the SCN cycle, which does not have a precise 24 h period, gradually shifts out of phase with the light- ing cycle. For example, if rats are blind or are kept in constant darkness for several weeks, the increase in N-acetyltransferase activity may occur during either the light or the dark period.

Stimulatory signals from the SCN pass through central and peripheral neural struc- tures to the pineal gland 4.5. Pineal cells are stimulated by the release of norepinephrine from nerve processes ='7. This transmitter diffuses to the surface of the pineal cell where it occupies adrenergic receptors =. N-Acetyltransferase activity is controlled through the betaa subclass of adrenergic receptors. The transmitter-receptor interac- tion activates membrane-bound adenylate cyclase, which increases the production and intracellular levels of cyclic AMP ~. cAMP is believed to provide the sole intracellular link between norepinephrine and mechanisms which control changes in N-acetyltranferase activity 1.

Intracellular events leading to stimulation of N .acetyltransferase

cAMP rapidly hyperpolarizes the pinealocyte's membrane a, and this seems to be required for an increase in N-acetyltransferase activity 9. cAMP stimu- lation of N-acetyltransferase activity clearly involves induction =°-19. We do not know whether the N-acetyltransferase gene, or a gene involved in the regulation of N-acetyltransferase activity is expressed as a result of adrenergic-cAMP stimulation. In vi tro, cAMP also acts through an activation/inactivation mechanism to con- trol this enzyme. Maintenance of high enzyme activity requires continuous eleva- tion of cAMP concentrations TM-

Inactivation of N-acetyltransfera~ The rapid decrease in enzyme activity

when animals are exposed to light at night is caused by light acting through the retinal projection to the SCN (Fig. 3; Ref. 5) to terminate transmission of signals to the pineal gland. As a result, transmitter is no longer released in the pineal gland. Molecules of transmitter remaining in the space between the neural processes and the pineal cells are then either destroyed or sequestered by the neural processes. This causes a rapid decrease in the extracellular concentration of norepinephfine, leading to dissociation of norepinephrine from the betaa-adrenergic receptor, and a decrease in adenylate cyclase activity TM. The rapid decrease in adenylate cyclase activity leads to a fall in intracellular cAMP TM which

SEROTONIN (5-Hydroxytryptamine)

(pmoles/mg)

N-ACETYLTRANSFERASE (nmoles product/mg/hr)

HYDROXYINDOLE-0- METHYLTRANSFERASE (nmoles product/mg/hr)

2.0

300

100 3.00 L-

1.00 ~

,60 ~

.30

.10

10

u

1.0

.6

! i!!!i~iiiiiiiii ~ i ! ! ~iiiiiii!, ~%iiiiiiiiii:iiiiii!iii~i~l

99

N-ACETYLSEROTONIN (5-Hydroxy-N-acetyltryptamine)

(pmoles/mg)

30 -

MELATONIN (5-Methoxy-N-acetyltwptamine) 1 0 -

(pmoles/mg) 6

1200 1800 2400 0600 1200

TIME Fig. 2. Rhythms in indole metabolism in the rat pineal gland. The daily variations in the concentrations o f metabolites and activities o f enzyme have been abstracted from reports in the literature (from Ref. 2).

appears to result in an immediate decrease in N-acetyltransferase activity, apparently through an inactivation mechanism. This inactivation is not the result of an abrupt halt in the synthesis of the enzyme 13. Restimulation of the gland does not signif- icantly reactivate N-acetyltransferase; new protein synthesis is required TM. Inactivation is effectively irreversible.

Evidence of a role for disulfide exchange in the inactivation of N-acetyltransferase

We have proposed that serotonin N-acetyltransferase is inactivated through a mechanism involving a protein thiol:disul-

fide exchange, leading to the formation of an inactive mixed disulfide 14,15:

ENZ-SH~t~e) + XSSX ENZ-S-S-X(inactive) + XSH

In preparations of broken cells disulfides inactivate N-acetyltransferase. Cystamine, for example, causes a rapid inactivation of the enzyme whereas the reduced form of this compound, cysteamine, does not. Inac- tivation by cystamine is also enhanced at higher pH. This is presumably because the reactive form of the protein thiol group, the thiolate ion (XS-), is present in larger amounts at higher pH thus accelerating

100

II RE J rHYPOTHALAMIC EYE lit RETINO- I ",'R,CT

SUPRACHIASMATIC NUCLEUS

~ l ENTRAPMENT I

ENDOGENOUS l OSCILLATOR J

TRANS M,SS,ON

I Tp

PINEAL GLAND

T I B S - M a r c h 1 9 8 2

RETRO- : CHIASMATIC [ HYPOTHALAMUSI

1 MEDIAL FOREBRAIN BUNDLE

I RYPTOPHAN I HYDROXYLASE

5HTp

ASE MECHANISM METABOLISM

/ /'1 \ I I OXIDASE TRANSFERASE PROTEIN SYNTHESIS

TOH~AA 1 N-Ac5HT HYDROXYINDOLE-O- 1 METHYLTRANSFERASE =

t MELATONIN

ADENYLATE I BETA CYCLASE ADRENERGIC RECEPTOR

I RECEPTOR

SPINAL CORD

1 SUPERIOR CERVICAL GANGLION

1 NERVE TERMINAL

Fig. 3. A schematic representation of the neural control of the pineal gland. ME, norepinephrine; cyclic AMP, adenosine 3',5'-cyclic monophosphate; Tp, tryp- tophan; 5HTp,5-hydroxytryptophan; 5-HT, 5-hydroxytryptamine, serotonin; HIAA, 5-hydroxyindole acetic acid; HTOH, 5-hydroxytryptophol; N-Ac5-HT, N-acetylserotonin. The question marks indicate unproven hypotheses (from Ref. 1).

disulfide exchange. The cystamine- inactivated enzyme can be reactivated to a limited extent by treatment with DTT 14. xs.

Studies on intact cells are consistent with these findings. In dispersed pinealocytes stimulated by treatment with norepine- phrine or dibutyryl cAMP, the enzyme can be inactivated by adding cystamine to the cell suspension ~5. Apparently, conditions inside the cell permit inactivation of N-acetyltransferase to occur v/a disulfide exchange.

This type of inactivation may be related to an earlier observation that pineal N-acetyltransferase is highly unstable in broken cell preparations ae. Incubation of homogenates of induced glands leads to a 90% loss of enzyme activity within 15 min. Interestingly, inactivation can be blocked

by acetyl-CoA 18,17. The acetyl group appears to be of special importance because CoA is far less effective. AcetyI-CoA may protect against inactivation by acetylating a critical thiol group of the enzyme 15-17 to prevent the formation of the inactive mixed disulfide derivative of the enzyme.

Acetyl-CoA also appears to protect against inactivation o f the enzyme by cys- tamine TM. Thus, inactivation by either an endogenous or exogenous inactivator can be blocked by acetyl-CoA treatment. Therefore, we suspect that the endogenous inactivating compound is a disulfide.

Other evidence is consistent with this interpretation. For example, a number of thiols, including penicillamine, cysteamine and CoA protect against inactivation ~4.~6. They could have this action because they

form inactive mixed disulfides with an endogenous disulfide which inactivates N-acetyltranferase and in this way neutral- izes it.

Two important questions arise: fh'st, what is the identity of an inactivating disul- fide compound? and second, how is such an interaction between this compound and molecules of N-acetyltransferase control- led? Cystamine might be the inactivator since an enzyme system has been character- ized which can convert cysteamine to cys- tamine~L Another possibility is that the di- sulfide inactivating compound is a peptide because argimne vasotocin, a disulfide- containing peptide (originally thought to be present in the pineal gland) can inacti- vate N-acetyltmnsferase by disulfide exchange 14.15. Although, the presence of

TIBS - March 1982

arginine vasotocin in the pineal gland is now in serious doubP a.2~, we suspected other disulfide-containing peptides could participate in the inactivation of N-acetyltransferase. Accordingly, we examined the effects of several disulfide- containing peptides ==. These included vas- opressin, oxytocin, insulin, pressinoic acid and several types of somatostatin. The most potent was insulin. These findings showed that the interaction of this protein with a disulfide-containing peptide is strongly influenced by the primary structure of the peptide. To our surprise, we also dis- covered that the most potent peptide inac- tivator of N-acetyltransferase, insulin, had essentially no effect on acetyl-CoA hy- drolase 2~ another pineal enzyme, which is activated by disulfide exchange 24. Somatostatin, was the most potent peptide activator of acetyl-CoA hydrolase. Thus, the reaction of a disulfide peptide with a protein thiol appears to be influenced not only by the primary structure of the disul- fide, but also by the structure of the protein; this is analogous to the relation of peptide hormones to their receptors, and substrates to enzymes.

How a protein thiol:disulfide exchange reaction could be regulated may eventually be answered by enzymatic or non- enzymatic explanations. This interaction might be catalyzed by a specific thiol trans- ferase 2~, perhaps controlled by cAMP. A non-enzymatic regulatory mechanism could be based on shifts in cytosolic pH because changes in pH can alter the rate at which disulfide exchange takes place. In addition, any shift in the redox state of an inactivating disulfide compound could severely alter its potency. For example, reduction of insulin blocks its capacity to inactivate N-acetyltransferase2L

A theoretical model of inactivation of N-acetyltransferase

A model to explain the inactivation of N-acetyltransferase via disulfide exchange, in which the enzyme exists in four forms is given in Fig. 415. The fast two forms, Form I and Form If, reflect the acetyl group trans- fer function of this enzyme. Studies using liver N-acetyltransferase show this enzyme exists as an acetylated intermediate, and it seems likely that this is true for the pineal enzyme. Following transfer of the acetyl group to an amine acceptor, the enzyme would then be in the free thiol form, Form II. In the acetylated state, Form I, the enzyme would be resistant to attack by di- sulfide inactivating compounds. However, in Form II, the free thiol could undergo di- sulfide exchange with an inactivating disul- fide. This would yield Form IlI, a revers-

101

I Active Thioacetyl 1 I -s-Ac CoA - - ~ t ' aCHzCHzNH2

AcCoA ~ RCHzCHzN HAc ,1 Active Thiol ! ~-SH

R,SHDT R,S-I, ~. = R,S"

III Inactive(Reversible)Mixed Disulfide I I "s-sR'

1: !

IV Inactive (Irreversiblel ~-s-? !

Fig. 4. A theoretical model of the inactivation of pineal serotonin N.acetyhransferase by disulfide exchange (from Ref. 15).

ibly inactive mixed disulfide. We suspect that Form Ill is transient and is rapidly con- verted perhaps via folding, to Form IV. This form is resistant to reactivation by reducing treatments. We base this on our general lack of success in attempts to reac- tivate N-acetyltransferase inactivated by in viva physiological mechanisms, and by mostin vitro treatments that cause inactiva- tion.

Purification of N-acetyltransferase vm protein thiol:disulfide exchange

It is interesting that the disulfide exchange can be used to purify pineal N-acetyltransferase TM . Cystantine is immobilized by attachment to Sepharose and enzyme preparations are passed over the column. The enzyme remains bound, presumably as a result of disulfide exchange. After the column is washed, active enzyme is released by DTT treat- ment. This procedure, combined with others, provides partially purified prepara- tions of the enzyme. These will be helpful in examining the molecular mechanism involved in regulating the enzyme.

General eonmaents The idea that protein thiol:disulfide

exchange is involved in regulation was fast proposed about 30 years ago 28 but it has not yet been clearly implicated in the physiological control of any enzyme. Perhaps N-acetyltransferase will be the fast. Furthermore, the wide use of DTr and other reducing agents as protectors against inactivation of enzymes, suggests that other enzymes may be controlled by a similar mechanism. Consistent with this line of

thinking, it also seems useful to consider that a family of intracellular disulfide- containing regulatory peptides may exist, each member of which participates in the selective regulation of a specific enzyme via disulfide exchange. This type of reg- ulatory mechanism could play a fundamen- tal role in biological systems.

References I Reppert, S. M and Klein, D. C. (1980,1 in Endo-

crine Functions o f the Brain (Maria, M., ed.), pp. 327-371, Raven Press, New York

2 Klein, D. C. and Namboodiri, M. A. A. ( 1981 ) in Monoamine Enzymes (Weiner, N., Usdin, E. and Youdim, M., eds) (in press)

3 Klein, D. C. and Weller, J. L. (1970) Science 169, 1093-1095

4 Klein, D. C., Weller, J. L. and Moore, R. Y. (1971) Proc. Natl Acad. Sci. U.S.A. 68, 3107-3110

5 Klein, D. C. and Moore, R. Y. (1979) Brain Res. 174, 245-262

6 Klein, D. C. andWelleL J. L. (1972)Scieme 177, 532-533

7 Brownstein, M. and Axelrod. J. (1974)Science 184, 173-175

8 Slrada, S., Klein, D. C.. Weller, J. L and Weiss. B. (1972) Endocrinol. 90, 1470-1476

9 Parfitt, A., Weller, J. L., Sakai, K. K., Marks, B. H. and Klein, D. C. (1975)Mol. Pharmacol. l 1,241-245

10 Klein, D. C., Berg, G. R. and Weller, J. L. (1970) Science 168, 979-980

11 Romero, J. A., Zatz, M. and Axelrod, J. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 2107-2111

12 Klein, D. C., Buda, M., Kapoor, C. L. and Krishna, G. (1978)Science 199, 309-31 I

13 Deguchi, T. and Axelrod, J. (1972) Proc. Natl Acad. Sci. U.S.A. 69, 2547-2550

14 Namboodiri, M. A. A., Weller, J. L. and Klein, D. C. (1980)Z Biol. Chem. 255, 6032-6035

15 Klein, D. C., Weller, J. L., Auerbach, D. and Namboodiri, M. A. A. (1980)inNeurotransmit- ters and Enzymes in Mental Diseases (Usdin, E.

102

and Youdim, Y. H. M., eds), pp. 60~627, John Wiley and Sons Ltd.. Chicbester, Sussex

16 Binkley, S., Klein, D. C. and Weller, J. L. (1976) J. Neurochem. 26, 51-55

17 Namboodiri, M. A. A., Nakai. C. and Klein, D. C. (1979)J. Neurochem. 33,807-810

18 Numboodiri, M. A. A. and Klein, D. C. (unpub- lished results)

19 Ziegler, D. M. (1980) Enzymatic Basis of Deox-

ification 1,201-227 20 Dogterom. J., Snijdewint, F. G. M., Pevet, P. and

Buijs, R. M. (1979) in Progress in Brain Research (Kappers, J. A. and Pevet, P., eds), Vol. 52, pp. 465-470, Elsevier/North-Holland Biomedical Press, New York

21 Negro-Vilar, A., Sanchez-Franco, F., Kwiat- kawski, M. and Sanisan, W. K. (1979)Brain Res. Bull. 4, 789-792

T I B S - March 1982

22 Namboodiri, M. A. A., Favilla, J. T. and Klein, D. C. (1981)Science 213,571-573

23 Namboodiri, M. A. A., Favilla, J. T. and Klein, D. C. (1981)Fed. Proc. 40, 1828

24 Naboodiri, M. A.,A., Favilla, J. T. and Klein, D. C. (1979) BBRC 91, 1166-1173

25 Freedman, R. B. (1979)FEBSLett. 97,201-210 26 Bah'on, E. S. (1951)Adv. Enzymol. Relat. Area

MoL Biol. 11,201-266

The slow-binding and slow, tight-binding inhibition of

enzyme-catalysed reactions John F. Morrison

lnhibi tors o f enzyme-cata lysed reactions can be div ided into f o u r classes according to the rate and strength o f their interactions with enzymes .

Enzyme inhibitors have played an impor- tant part in advancing our knowledge of biochemistry. This is especially true for those inhibitors which function as substrate analogues and have been used to elucidate metabolic pathways and kinetic mechan- isms of enzyme-catalysed reactions. Most studies, with reversible inhibitory substrate analogues have been performed under steady-state conditions where the concen- tration of enzyme is very much less than that of the inhibitor (and substrate) and where all the equilibria involving the enzyme and reactants are set up rapidly. However, there are inhibitors that do not satisfy one or either of these two conditions.

As far back as 1943, it was recognized that there are compounds which inhibit enzyme-catalysed reactions at concentra- tions comparable to that of the enzyme and under conditions where the equilibria are set up rapidly 1. Such compounds are refer- red to as tight-binding inhibitors. For the derivation of rate equations that describe tight-binding inhibition, allowance must be made for the reduction in the inhibitor con- centration (It) that occurs on formation of an enzyme-inhibitor (El) complex. No longer does the fh'st steady-state assump- tion hold. The concentration of EI is not negligibly small compared with It and the free concentration of inhibitor is not equal to its added concentration. The resulting equation, which is a quadratic function with squared and linear terms in velocity, pre-

John F. Morrison is at the Department of Biochemistry, Australian National University, Canberra, A CT, Australia.

dicts that plots of velocity against total enzyme concentration (E,) at different total inhibitor concentrations (It) will be cur- vilinear 1. It also predicts that a double- reciprocal plot of velocity against substrate concentration in the presence of the inhibitor will have curved and linear por- tions. For competitive inhibition all the lines will converge at a common point on the vertical ordinate. However, unless rela- tively high concentrations of substrate are used, the curvature of the plots may be mis- sed. Thus, a compound which might be expected to act as a linear competitive inhibitor with respect to the variable sub- strate could appear to act as a non- competitive inhibitor. Such a misinterpreta- tion can be avoided if one recognizes that, with tight-binding inhibition, the slopes of the lines of a double-reciprocal plot do not vary as a linear function o f l ? .

As the strength of interaction between an enzyme and a tight-binding inhibitor increases, a stage is reached when the equilibrium of the reaction cannot be estab- lished rapidly 2.

For the reaction E ~ El k2

the apparent first-order rate constant (k uS) will be small when only a very low concen- tration of L is required to demonstrate the inhibition. The slow establishment of the full inhibition of enzymes has been long known and frequently circumvented by pre-incubating the enzyme with the inhibitor before the addition of substrate.

Over the past few years it has become

© E l ~ v i c r B i o m e d i c a l Press t 9 8 2 0 3 7 6 - 5067/82/CO00 - ~ / $ 0 2 . 7 5

clear that the slow establishment of equilib- rium for the reaction of an enzyme with a competitive inhibitor can occur either with or without tight-binding inhibition 3. Indeed, it seems that inhibitory substrate analogues can be grouped into four categories on the basis of the relative con- centrations of inhibitor and enzyme and whether the equilibria are established rapidly or slowly (Table I). Little has been written about slow-binding inhibition 4 which is the subject of this article. It will be assumed that all slow-binding inhibitors give rise to reversible, competitive inhibi- tion.

Slow-binding inhibition

When a reaction, in the presence of a slow-binding inhibitor, is started by the addition of enzyme the relatively rapid ini- tial velocity decreases to a slower steady- state rate (Fig. 1). If the enzyme is pre- incubated with inhibitor and the reaction started with substrate, there will be a slow release of inhibition and ultimately a steady-state rate will be reached. The two steady-state rates will be identical provided that there is no significant enzyme inactiva- tion, substrate depletion or product inhibi- tion. It should be noted that slow-binding inhibition is, in effect, transient-state kin- etics on a steady-state time scale. While in- itial and steady-state rates are considered to be synonymous for steady-state kinetics studies, the two rates are quite distinct for slow-binding inhibition.

When the concentration of inhibitor is ten or more times greater than that of the

TABLE I. Classification of reversible enzyme inhibitors

Characteristics Rate of

establishment of Relationship equilibrium

Class of between between inhibitor Et and It E, I and El

Classical It :>>Et fast Tight-binding It ~ Et fast Slow-binding It ~>> Et slow Slow, tight-

binding It = Et slow