TIBS 19 - OCTOBER 1994 FRONTLINES

Invasion of the cabbage patch* (should biochemists learn the Krebs cycle or the cell cycle?)

What is Biochemistry? This question may be irrelevant for practitioners, but it haunts the teachers, course organizers, textbook authors and editors of maga- zines that contain the word in their titles. In his essay composed in 1937 for Sir Frederick Gowland Hopkins' festschrift ~, the geneticist J. B. S. Haldane opened with a familiar and perhaps surprisingly conventional view: 'The ultimate aim of biochemistry may be stated as a complete account of intermediary metabolism, that is to say, of the transformations undergone by matter in passing through organisms', although by the end of the piece he was presciently saying: 'Gene reproduction is...a task for the biochemist'. Joseph Needham's bold credo in the same vol- ume is more striking. He begins: 'For the biochemist, the problem of organic form is ultimately unavoidable'. Perhaps Rudolph Peters, who used to rush up and down stairs while well into his 70s, carrying vials of radioactive fluorine-18 (the half life of which is 11.2 minutes) fresh from Harwell, got closest to the heart of the matter when he mused ' . . .whether there is any need for specialized biochemical research. Should the subject now go back again to chemistry and physiology?'L But this would be a disaster, he concluded, because biochemists are paid to 'think about the chemistry of living matter', implying that if they didn't, then nobody would.

It is by turns inspiring and amusing to read the 57-year old thoughts of these distinguished disciples (at least two Nobel prizewinners among them) of Sir Frederick Gowland Hopkins, the great man who, at the age of 53, founded one of the first actual biochemistry depart- ments, in Cambridge in 1914. These men and women had complete faith that it would one day be possible to explain how living organisms worked in chemical terms. As it turned out, it was to be sooner than they thought. Biochemistry has been a triumphant success. Paradoxically, perhaps, the very success of biochemistry has made

© 1994, Elsevier Science Ltd 0968- 0004/94/$07.00

it no easier to answer the question: does biochemistry really exist? And if so, what, precisely, does it comprise? John Gurdon once told me that it wasn't a real subject, just a collection of techniques, a view shared in part by Tim Mitchison, who tells me that he recently taught an introductory course at UCSF that dealt with solutions, pH, dissociation constants and column chromatography. Apparently, these dry subjects proved highly popular, even in competition with the latest stuff on cell- cycle control and the molecular biology of development. Nobody had thought to teach this fuddy-duddy material for so long that it was fresh again (and never more relevant, one might add). This sent me back to my own undergraduate notes, Part II Biochemistry, University of Cambridge, summer of 1963: sure enough, we were taught thermody- namics (not very well), the theory of column chromatography, immuno- chemistry and microbiology; and on July 26 Carl Cori told us about the control of phosphorylase by reversible protein phosphorylation, and the sem- inar must have been compelling, because ! even typed up my notes!

Those were happy days, when the workings of the mitochondrion were still a mystery; the genetic code had only one letter; the structure of myo- globin and haemoglobin were just beginning to sharpen enough to reveal individual amino acids; and metabolic feedback control was magically fresh, explaining how bags of enzymes could have reasonably coherent inputs and outputs. The hard core of biochemistry now comprised the metabolic path- ways, their energetics and their con- trol. DNA, RNA and protein synlfhesis were slightly naughty subjects that we read about after lights-out, so to speak. Our excellent but very dated textbook was Ernest Baldwin's Dynamic Aspects of Biochemistry2; Lehninger and Stryer were not even specks on the horizon.

All this has changed. Metabolism, its control and its disorders have by now

practically attained the status of Latin, a dead subject in all but a few ancient, marble-topped, mahogany-clad temples, but knowledge of which is, for some all-but-forgotten reason, required to get into medical school. The tension is between Haldane and Needham, between the Krebs cycle and the cell cycle, so to speak. Which (if either) should you learn about? Enzyme struc- tures and mechanisms are studied in at least as many departments of chemistry as biochemistry, and might well be better treated in the former. Devel- opmental biologists use biochemi- cal approaches to make their great strides, and even geneticists are rec- onciled to molecules. Biochemistry has been so successful that it now en- compasses everything and scarcely exists as a coherent 'subject' any more. Many a biochemistry department houses a range of workers from physicists to developmental geneticists, which would doubtless gladden the heart of Sir Frederick: the only problem is, their only common language is the genetic code. And the students of my acquaint- ance who come to biochemistry to understand developmental neurobi- ology do not want to hear about recipro- cal space, nor (I would argue) do they need to know how X-ray crystallography works, despite its extraordinary revelations.

Sad to say, these considerations dc not in the least help designers of under- graduate courses or writers of text- books. Something of the old curriculum must perforce be lost for every new dis- covery that comes in. And if we used to mock the rote learning of the Boehringer metabolic wall chart, the map of on- cogenes and signal/transduction path- ways is really no more edifying. ! used to look with despair at the books and papers we were supposed to read and learn (understanding was quite another matter): 'what must it be like for a beginner today?

We like to think that TIBS provides a natural forum for the discussion of these issues, and would welcome cor- respondence from the designers of introductory biochemistry and molecu- lar biology courses for biologists and medical students. What is biochem- istry at the close of the century? Where is it heading? Some of my friends

*'If biochemists were not so tremendously keen to see their labours rapidly extended by borderline specialists, they might be found to view with annoyance the invasion of their cabbage patch.' R. A. Peters 1.

395

TALKING POINT consider that a more chemical approach is desirable, as more and more structures are solved and fasci- nating mechanisms are revealed; but I myself think, along with Sir Rudolph Peters, that what my generation were trained to be were 'molecular physio- logists' above all else. For, as Peters wrote, 'it would hardly be too frivolous to suggest that the best definition of

biochemistry to-day is that it is the part of chemistry called "physiology" by the pure chemists and "chemistry" by the pure physiologists. ' Of course the important thing, really, is to get on and do it, and not to worry about names. But please let us know what you think.

~geferences 1 Perspectives in Biochemistry (1937)

TIBS 19 - OCTOBER 1994

(Needham, J. and Green, D. E., eds), Cambridge University Press

2 Baldwin, E. (1963) Dynamic Aspects of Biochemistry (4th edn}, Cambridge University Press

TIM HUNT

ICRF Clare Hall Laboratories, South Mimms, Herts, UK EN6 3LD. emaih t..hunt@icrf.icnet.uk

An allosteric transitioi~ is a change in the binding or catalytic properties of a molecule brought about by the binding of an effector molecule at a site distinct from that which confers the binding or catalytic property. This month's Talking Point discusses unprecedented evidence from the nicotinic acetylcholine receptor that demonstrates a clear switch between conformational states with or without ligand binding, suggesting that these states pre-exist, in support of the Monod-Wyman-Changeux allosteric model.

w

Single channel currents in the nicotinic acetylcholine receptor: a direct demonstration of allosteric

transitions

The so-called allosteric theory of Monod, Wyman and Changeux at. tempted to explain how small ligands regulate protein function ~, This theory has described a wide range of exper- imental phenomena with remarkable success z,2. At the heart of altosteric the- ory is an essential assumption that the protein can interconvert or isomerize between two stable conformations or states. This interconversion was re- ferred to as an allosteric transition ~, but its existence has often been viewed with skepticism s. In the book Protein Interactions 4, G. Weber cited a lack of direct experimental support for such protein isomers in solution and went on to claim that 'over these many years the proof of the coexistence of these con- formations has not been forthcoming.' The position offered here is that the hypothesis of allosteric transitions has been verified directly for the nicotinic acetylcholine receptor (nAChR) by experiments with the patch-clamp

396

technique. The nAChR emerges as a very well-understood allosteric protein, and its activation mechanism serves to illustrate some key principles of the M.onod-Wyman-Changeux theory.

How might Iigands regulate proteins? Skepticism regarding the existence of

allosteric transitions is rooted in the evi- dent faUacy of describing a protein in terms of just a few stable states. This flies in the face of the complex stochastic conformational dynamics of proteins, and ignores a protein's thou- sands of internal degrees of freedom. Furthermore, the success of allosteric theory in describing many experimental results does not validate the underlying assumptions of this theory, because other theories based on distributions among a continuum of states can often reduce to simple two-state or few-state representations ~. On the other hand, the postulate of a concerted ailosteric

transition between very different stable tertiary structures provides an elegant solution to the problem of how several functions and properties of a protein change concomitantly. The hypothesis can be implemented relatively easily in theoretical formulations: binding energies add linearly, and are taken as mutually independent within each conformationa[ state ~, The simul. taneous change in all of the binding sites, presumed to occur during an aliosteric transition, accomplishes the task of linking the state of occupancy of each binding site to the others. Alternatives to linkage between binding sites via allosteric transitions include the transmission of binding energy through long-range molecular forces, and long-range deformations of the protein originating at the binding site s. A model invoking sequential conformational transitions within individual subunits s represents an inter- mediate form of linkage between bind- ing sites that falls between the two extreme cases of a global allosteric transition of the entire protein and deformation originating at the binding site. Can we distinguish between these strikingly different views of how ligands regulate proteins? Evidence for the existence of allosteric transitions would help.

Direct evidence from the nAChR The patch-clamp technique, which

can monitor the current through single ion channels on t imescales in the

© 1994, Elsevier Science Ltd 0968- 0004/94/$07.00