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Sci Tech
Proline — the world's smallest enzyme
WE ARE always fascinated to know about the world's smallest or biggest things. Here is one; what is the smallest drug in the world? My answer is the lithium ion. Just a fraction of a nanometre in size (even including the inevitable water molecules that cling to it in aqueous solution), it must be the prize- winner. It's salts (chloride, iodide) are used as drugs against manic depression.
Now consider the tiny germ mycoplasma genitalium. It has a genome containing but 470 genes. Drs. Craig Venter and his wife Claire Fraser asked how many of these genes are vital, how many redundant, and what is the minimum number of genes with which the bug can carry on living. To this end, they progressively removed gene after gene from this organism and tested its ability to live. They found that it can make do with just 320 genes. This truncated version of M. genitalium holds the world's record of having the smallest genome of any living organism. (Viruses with smaller genomes cannot compete since they are not living. To be called living, you must be able to do two things: reproduce and metabolise. Viruses can only reproduce, so they are but half-alive).
Incidentally, this Venter - Fraser experiment gives me the vain but tempting opportunity to boast a bit. While discussing their experiment in my column of March 11, 1999, I had claimed that it is well within our capability today to chemically synthesize this 320-genes-long genome of M. genitalium in the lab, insert it in a suitable artificial `cell' and kick-start life. I had then boldly, if rashly, predicted that such creation of life by man may happen well before 2020. Well, Dr. Venter has declared last month that his group has started this experiment with the truncated M. genitalium. I wait with bated breath.
What is the smallest gene? I am not sure if we know the answer. There are several claimants and I welcome mail from readers on this issue; likewise on the smallest protein. I suspect, though, that the moment these two are announced from the natural world, chemists will get busy chipping them away to create even smaller candidates, just as Venter did with the genome.
It is in the same spirit that a claim has been made for the world's smallest enzyme. Writing in the 6 December 2002 issue of Science, Drs. Mohammed Movassaghi and Eric N. Jacobsen of Harvard University have suggested that the amino acid proline may well qualify for the title of the simplest enzyme. Being less than a nanometre in size, it qualifies to be the smallest as well. The Harvard scientists cite several organic chemical reactions, belonging to the aldol addition class, where the presence of proline speeds up the reaction and also helps generate products of a single handedness.
Is this a play on words or does proline qualify? What is the difference between an enzyme and a simple catalyst? Does any molecule that enters into and exits from a reaction cycle qualify to be called an enzyme? These are some questions that purists will raise. In answering these, we need to stay clear of terms used by tradition and look into the actual roles played by the molecules involved. The term enzyme was coined by Emil Fischer and others over a century ago for substances that helped fermentation reactions (the word zyme is a learned borrowing from Greek for fermenting or leavening). It was later found that just about every enzyme molecule was a protein. The mechanism of enzyme action was unravelled to be catalytic, using the large surface that the enzyme molecule provides.
Typically, the protein molecule that acts as the enzyme offers sites of specific shape and geometry into which the reacting molecules fit. The reactants, so accommodated next to one another on the enzyme surface, chemically react to generate the products. The enzyme protein molecule helps the process by offering proximally placed groups of atoms from its own structure. Once the reaction is over, it takes back these loaned groups and regains its original chemical identity, so that it is ready for the next cycle of reaction. At the same time, it also releases the products off its surface so that it is ready to receive the next pair of reactants.
Dissecting the whole reaction cycle, we note three features basic to enzymatic catalysis, namely specificity, speed and turnover. First is the binding or capture of the reactants on the protein surface. There is a built-in specificity to this. The actual shape — the crests, crevices and pockets — that the polymeric enzyme molecule offers, accommodate only form-fitting molecules. This is the famous lock and key fit, or the hand and glove fit which biological polymers are famous for. The enzyme trypsin can accommodate only the shape of lysine-type amino acids in its `active site' or `binding site' and catalyse the hydrolysis of the peptide group adjacent to this lysine. It does so by offering its own reactive groups at the `catalytic site', and taking them back at the end of the reaction. The next feature is the speeding up of the reaction.
Had the enzyme not bound the reactants and placed them next to each other and helped in the reaction process, the reactants would have had to diffuse around the solution finding each other — a far slower process, and an uphill task. The enzyme makes this hill-climbing far easier through its proximal binding of the reactants and offering itself as the go between. During this process, all partners in the reaction are pre-arranged at the `ready, steady, go' position on the enzyme surface. The transition state between the start and the end of the reaction has been attained. The last feature is the jettisoning of the products from the enzyme surface, and regaining the groups it lent, and also its original shape and surface. This is the turnover stage. An enzyme can typically run through thousands of cycles of the reaction in a second, and convert gram quantities of reactants into products in a minute.
Enzymatic ability need not be restricted to proteins alone. In principle, any macromolecule with a shape, binding surface and reactive groups should act as an enzyme. Sure enough, Sidney Altman and Thomas Cech showed this to be true of nucleic acids, RNA in particular. This has led to the suggestion that life started on earth perhaps in the RNA — both as the genetic molecule coding for replication and as a catalytic molecule helping in metabolism. More recently, even DNA (single strands in particular) has been shown to be enzymatic in character. Among the people to design ribozymes and DNAzymes is our own Akhil Banerjee of NII, New Delhi.
The review by Movassaghi and Jacobsen on the role of the amino acid proline as an enzyme removes even the need for an enzyme to be a polymer with a large surface. Indeed one of the first reports of proline as an enzyme was made by the group of Richard Lerner, at the Scripps Institute in California. Though there were earlier reports, it was the paper in the year 2000 by B. List, R. Lerner and C.F. Barbas III in the Journal of the American Chemical Society on proline catalysing the aldol condensation of the acetone to various aldehydes with high stereo-specificity (proline as an aldolase) that has set the pace.
Till then, small molecule catalysts used for such purposes involved organometallic complexes of artificial creation. Proline, on the other hand, is a natural, having been on earth well before 3-4 billion years. It may thus well be the smallest natural enzyme; unless one finds azetidine carboxylic acid, which occurs in the lily- of- the- valley, too can do so. It is not clear whether the even smaller cousin of proline, aziridine carboxylic acid, occurs in nature. Professor Bhaskar Maiya of the University of Hyderabad, whom I consulted, suspects that it does not, but that there are reports that it actually inhibits enzymes — `anti-enzyme'!
D. Balasubramanian
dbala@lvpeye.stph.net
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