RESEARCH >
N e w Theory Explains Enzyme Action " I n d u c e d fit" theory o f e n z y m e specificity questions the old " t e m p l a t e " t h e o r y c a n ' t
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There's new NATIONAL progress in unMEETING raveling the mystery of how enBiological zymes work. These Chemistry vital chemicals can distinguish between closely similar chemical structures. How isn't fully known yet. Theories fit most situations but some have defied answers. A ne^v "induced fit" theory of enzyme specificity seems to provide answers for aLl situations run into so far, Daniel E . Koshland, Jr., of Brookhaven National Laboratory told t h e Division of Biological Chemistry. Until now, enzyme specificity was explained by t h e "template" ox "key-lock" theory proposed b y Fischer and others in t h e 1890's. An enzynxe was considered more or less a rigid template or negative with attractive amd repulsive regions arranged to fit desired substrates and repel compounds that differ even in only minor ways. Sort of as a key fits a lock. Glucose, for example, is a substrate for the enzyme hexokinase. But a d d a methyl group at position J5 of glucose, a n d it's no longer a substrate. I t ' s too big to fit the template, says Koshland. Similarly, even a minor chemical c h a n g e such as going from a methyl group to an ethyl group ixi some substrates prevents reaction -with a n enzyme because ethyl is too large for the mold. • Holes in t h e Theory. But there are weak spots in the template theory. A glaring one, says K o s h k n d , is a situation where smaller analogous comp o u n d s fail to react. For example, replacing a hydroxyl group w i t h a hydrogen results in a smallea: compound w h i c h should fit t h e template and therefore react. T h e smaller comp o u n d s often get on the enzyme surface; but they don't react. W o r k with isotopes h a s uncovered several other similar situations, says Koshland: 60
AC$
C&EN
SEPT.
23,
1957
answers
• Phosphotransacetylase reacts w i t h acetic, propionic, and butyric but not with t h e smaller but similar formic group. • Beta glucosidase acts on glucoside but not on 2-desoxyglucosides.
> N e w T h e o r y . This is where Koshland's "induced fit" theory comes in. Instead of a rigid template, t h e enzyme is thought of a s a flexible macromolecule a n d not a perfect negative of t h e substrate a t first. Here's how it works: As the substrate approaches t h e enzyme it induces a change in the coiling of the protein, the various attracting and buttressing groups aligning t h e m selves properly to form a complementary structure w i t h the enzyme. W i t h t h e p r o p e r substrate, the catalytic group of the enzyme is in the right place relative t o the substrate b o n d t o b e broken so t h e reaction can proceed. The catalytic group falls into t h e right p l a c e only if the c o m p o u n d has t h e r i g h t size and proper attractive groups. If an attractive group is miss-
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ing, a link on the enzyme isn't held in place. This skews the reactive group out of position and no reaction occurs, even though the substrate is the same size as the proper one. Leaving out a group so the substrate is smaller than the right one also causes misalignment. The smaller chemical can be absorbed on the enzyme surface but it won't react because the reactive part isn't in the right place. The new theory, points out Koshland, incorporates features of the template theory—in both, die substrate must fit. The big difference: In the "induced fit" theory, the fit occurs only after changes in the enzyme structure. Actually, says Koshland, "We're only on the first lap of a fairly long run" in getting a complete picture of how enzymes work. But a clearer understanding of the way enzymes achieve specificity will help explain how living systems are regulated. Then, too, the door may be opened t o some real progress in chemical theory. No man-made catalyst, says Koshland, has been able to duplicate an enzyme's specificity.
Rotation and NMR N M R detects bond d i f f e r ences in isomers, allows c a l culation of barriers t o internal rotation
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ACS NATIONAL
MEETING Physical & Inorganic Chemistry
When
chemists
learn enough about x.1.
i -
the inner workings of known molecules, they can feel more sure-footed about extending what they know to more complex molecules and, indeed, to predicting properties of molecules as yet unknown. These ideas are a major driving force behind all spectroscopy and, specifically, behind a nuclear magnetic resonance program that D u Pont has been pressing for the past four years, according to W . D . Phillips. One subject the D u Pont people are probing is hindered rotation in molecules. The two general aims: • To learn what the facts shown by N M R mean in terms of hindered rotation. • To learn to relate these NMR data to energy barriers to internal rotation, and to things like molecular geometry and electronic structure. NMR has proved extremely useful in
this • work because it can distinguish between the different types of bonds present in different stereoisomers of a particular molecule. But if the isomers switch from one form to the other faster than roughly 1000 times per second, NMR won't show discrete peaks, Phillips told the Symposium on Nuclear Magnetic Resonance Spectroscopy, sponsored jointly by the Organic and the Physical and Inorganic Divisions. Sometimes this problem can be evaded by cooling the system, thus slowing the rate of reorientation. It turns out, says Phillips, that this temperature transition point (in terms of whether NMR works or not) can be pinned down quite accurately. And by studying NMR spectra around that point, one can figure out the energy barriers to rotation about bonds. Sometimes, as with nitrosamines, the system must be heated to get it close enough to the transition point to do this. • It's Partly Double. In nitrosamines rotation is hindered around the N—N bond, which has long been written as a single bond. Hindrance around a single bond should be low, but NMR shows that it's quite high. This means, says Phillips, that the bond behaves, in part, like a double bond—i.e., dislocated electrons result in pi bonding, as in benzene. NMR also shows such a bond (O—N) in alkyl nitrites. Here, NMR can show the temperature dependence of the cis/trans isomer ratio, and thus the energy difference between the two forms. It comes out to be rather low, confirming that the barrier to rotation around the O—N bond is due to its partial double bond nature, rather than to hydrogen bonding, as had been assumed. In oximes, NMR shows hindered rotation about the C—N bond as expected; it's written classically as a double bond. But in oximes of straight chain aldehydes the syn/anti (analogous to cis/trans) isomer ratio is close to one, and the anti form actually predominates in acetaldoxime. This was unexpected, says Phillips, because the alkyl substituent is much bulkier than the aldehyde proton. However, certain branched aliphatic oximes seem to conform more closely to the expected pattern. NMR will detect thermal interconversion of isomeric oximes; D u Pont is working on quantitative values for the energy barrier to rotation about the C - N bond. Ethanes have, in general, three isomeric forms. But barriers (van der Waals forces) to rotation around the
C—C bond are quite low, so the isomers switch back and forth too fast for NMR. In a few cases, says Phillips, they've managed to whip this problem Ly dropping the temperature. Below —80° C , for instance, NMR shows two rotational isomers of BrF 2 C—CBr 2 CN. The asymmetrical form seems about twice as populous as the symmetrical form; the energy difference between the two is relatively small, the asymmetrical form the more stable.
Solid Monolayer Films Lubricate Adsorbed monornolecular polar compounds a r e keys to good lubricity a n d w e a r prevention
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A thin, protec-
ACS
NATIONAL _ _._ _AAPFTIMG Colloid.
tive film of a chemi c a i addition agent is the key to good Chemistry lubrication. William A. Zisman and coworkers at the U. S. Naval Research Laboratory in Washington affirm this and say that such a coating should consist of polar molecules adsorbed as solid monolayers on rubbing surfaces. New surface chemical methods developed at NRL permit adsorption of a well-defined, closely packed, monomolecular film of any adsorbable compound and isolate this coating on the surface of metals or other smooth solids. Measurement and control of molecular packing in condensed monolayers are obtained by measuring contact angle, or wettability, of a reference liquid— methylene iodide—on a film-covered surface. NRL finds now that structure and constitution of these films is indicated by variation in coefficient of friction when lubricated surfaces continue to rub together. • Sliding-Bal! Tester Used. By sliding a polished steel ball subjected to known pressure over the same path repeatedly on a flat, lubricated surface of metal or glass, the coefficient of friction can be recorded continuously, Zisman's group told the Division of Colloid Chemistry. Also, this method is a powerful research tool for studying durability and wear-prevention of adsorbed films, they feel. On the basis of significant differences in frictional and durability behavior of adsorbed monolayers, NRL groups the many types of films studied into three SEPT.
2 3,
1957
C&EN
61
