contemporcwy hi~tory
LEONARD editedFINE by: Columbia University New York. NY 10027 ERICS. PROSKAUER
The Application of Physical Organic Chemistry to Biochemical Problems Frank Westheimer Haward University, Cambridge, MA 02138
The application of the concepts of physical organic chemistry to the understanding of the mechanisms of enzyme action constitutes one of the maior triumnhs of our science. The principles pioneered by Lapworth, Ingold, Hammett, and others led. in a relativelv short neriod. to a revolutionarv change in the field of enzydo~ogy. i n 1941, no one knew thk mode of action of a single enzyme or coenzyme; by 1963, however, the catalytic mechanisms were known for three coenzymes and for several enzymes that function without cofactors. The way was then opened to an understanding of enzymic mechanisms at nearly the same level as that that Hammett had established by 1940 for nonenzymic chemistry in his now classical text ( I ) . Although Hammett never carried out research in biochemistry, his text was almost as essential t o the development of mechanistic enzymology as to that of mechanistic organic chemistry. It is an honor for me to acknowledge his part, as my post-doctoral advisor, in my career. I shall, to the best of my ability, outline the significant events that made enzymology almost a branch of ~hvsicalorganic chemistrv. - This disc&ion will be ;estricted to some of the discoveries made nrior to 1963, that is, to work comnleted a t least 20 years ago. Although this distance should Govide me with a little perspective, I must begin with a disclaimer. History is a much more difficult subject than chemistry. The first mentions of specific ideas or the first examples of principles are often hard to find, and I am not certain that I have found them. But the first example may not he important anyway; what is important is the chemistry that excited others and stimulated further development. Not only is it difficult to determine what publications or lectures provided this essential stimulation, hut some of the important actors in the story are unfortunately no longer on the scene to correct any errors or misconceptions that may mar this essay. What I present here is offered with simultaneous conviction and diffidence. Before enzymology could he put on a mechanistic footing, enzymes had to he purified and their structures determined. In addition, the techniques of physical organic chemistry, such as reaction kinetics, spectroscopic identification of intermediates, the uses of isotopes, acid-base catalysis, and Presented at the Louis Hammen Symposium on the History of Physical Organic Chemistry at the 186th National Meeting of the American Chemical Society in Washington. DC. August 31, 1983. and extracted from chapter in Vo1 21 of "Advances in Physical Organic Chemistry" (Bethell. D.; Gold, V.. Eds.) with permission of Academic Press.
Bransted and Hammett relationships had to he developed. The purification and crystallization of proteins had been achieved between 1926 and 1935 hv Sumner. Northron. Kunitz, and others (Z), but the first step in establishing t f ~ d structures of nroteins. indeed the essential sten in showine that proteinshad strkctures, as conventional& defined b; oraanic chemists, was taken onlv in 1951 when Frederick sanger determined the amino acid sequence for insulin (3). The determination of the three-dimensional structure of proteins by X-ray analysis dates from the publications of John Kendrew ( 4 ) on myoglobin and Max Perutz (5) on hemoglobin in 1960. Thus the early advances in nnderstanding enzymic mechanisms occurred a t the same time as the establishment of protein structure. Regrettably, most organic chemists in the 1950's regarded enzymology as an unrewarding, if not an improper, field of investigation. For many years, synthetic organic chemists were captivated by the chemistry of sterols and alkaloids and physical organic chemists were concerned with solvolysis. The intellectual advances were stunning, but the result was to leave the field of mechanistic biochemistry largely to the biochemists. In the late 19th century, Emil Fischer, a card-carrying organic chemist, used maltase and emulsin to establish the stereochemistry of anomeric derivatives of sugars, and clearly regarded enzymology as part of his territory ( 6 ) .In his Nobel lecture in 1902, Fischer wrote, in part, "I foresee the day when physiological chemistry will not only make extensive use of the natural enzymes as agents, hut when i t will prepare synthetic ferments for its own purposes" (7). The application of the principles of physical organic chemistry to enzymology enlarges the knowledge base on which to build Fischer's synthetic ferments. I t is unlikely that he expected it to take us a century. Coenzyme Mechanisms The first coenzvme for which the mechanism of action was discovered was p;ridoxal phosphate, which functions in the transamination. racemization, and decarboxvlation of amino acids as well as in the dehydration of Herine and the svnthesis of trvptonhane. Pvridoxal, for which the structure was established in-1939, is bne of the three active forms of vitamin Bs. The requirement of this coenzyme for amino acid metabolism had been demonstrated by 1945, in part by the work of A. E. Braunsteio and his co-workers in Moscow (a),but the complete mechanism, showing the chemistry of action of the coenzyme, was not written out in detail until 1953. At that time, Braunstein and M. M. Shemyakin ofVolume 63 Number 5 May 1986
409
P
H-C-NH,
I
CH,OPO,H,
dideuteroethanol as a substrate, we demonstrated that one atom of deuterium is transferred from substrate to coenzyme, CH3CD20H+ NAD+ CH3CD0 + NADD (1)
-
+ O=CH
COOH R
R
I
COOH
Figure 1. Amino acid metabalism
fered their formulation, complete with the curved arrow notation of physical organic chemistry, as shown in Figure 1: the coenzyme acts as an electron sink in a ketimine mechanism. Initially, an amino acid reacts with pyridoxal phosphate to yield pyridoxamine phosphate and a p-ketoacid. The pyridoxamine then donates its amino group to another p-ketoacid by a reversal of the reactions shown. The mechanisms they advanced for transamination, racemization, and (with some help from M. Hanke and his collaborators (10)) for decarhoxylation were exactly what we would postulate today, and they constituted an early and successful application of theory to mechanistic enzymology. I t must be admitted, however, that the theory appealed because it was reasonable and not because the authors had compelling evidence in terms of physical organic chemistry to support their formulation. At the same time, E. E. Snell and his co-workers a t Berkeley reported results using model reactions between pyridoxa1 and glutamic acid, results that certainly carried an implication of mechanism (11). They, too, developed the modern mechanism for this series of reactions and demonstrated the role of the coenzyme as an electron sink by substituting alternative catalysts for pyridoxal phosphate. In particular, they showed that 2-hydroxy-4-nitrobenzaldehydefunctioned in their model system just as the vitamin does. Although this early work left unanswered all the questions concerning the role of the protein in accelerating the reaction, the basic mechanistic pathway had been developed in accordance with the electronic principles of physical organic chemistry and confirmed by studies on model systems. In the same period, Bergit Vennesland, Harvey Fisher, and I partially elucidated the chemistry of some enzymic oxidation-reduction reactions (12). We were concerned with the mechanism of action of the nicotinamide adenine dinucleotides, NAD+ and NADP+, which function as coenzymes in many of the important oxidation-reduction processes of metabolism. In particular, they serve in the oxidation of ethanol to acetaldehvde and in the reduction of ~ v r u v a t eto lactate. The questiod we set out to answer was w6ether these reactions occur hv direct transfer of hvdroeen - " from the substrate to the coe&tne and vice versa. An alternative path was an electron transfer between substrate and coenzyme, facilitated somehow by the enzyme, with exchange of hydrogen between the suhstrate and the solvent. The first system we studied was that of yeast alcohol dehydrogenase. Using 410
Journal of Chemical Education
and, further, that this atom of deuterium could then be transferred from the coenzyme under the influence of lactic dehydrogenase to pyruvate, CH,COC02H + NADD
-
CH3CDOHCOzH NADi
(2)
The converse experiment was also performed, that is, the reactions were carried out with normal substrate and coenzyme but in DzO; this resulted in the transfer of ordinary hydrogen from substrate to coenzyme. The question we had set out to answer was then settled. The oxidation-reduction proceeds by direct transfer of hydrogen between substrate and coenzyme. But the results provided an extra bonus. Deuterated reduced nicotinamide adenine dinucleotide, NADD, prepared by the enzymic reduction of NAD+ with dideuteroethanol, has both a hydrogen and a deuterium atom a t the 4 position of the dihydropyridine ring. In the reduction of pyruvate or acetaldehyde with this NADD, deuterium is quantitatively transferred from the coenzyme to the carbonyl compound, while the hydrogen atom remains in place. The carhon atom in the 4 position of NADD is clearly chiral. Furthermore, when acetaldehyde is reduced by NADD, the resulting monodeuteroethanol is also chiral, and consists of only one enantiomer. When the monodeuteroethanol is enzymically reoxidized by NADf, only the deuterium is transferred from the alcohol to the coenzyme (eq 31, CH,CHO
+
NADD
=
OH D+H
+
NAD+
(3)
+
NAD+
(4)
CHa
CH,CDO
+
NADH
OH H+D
CHa Conversely, when deuteroacetaldehyde is reduced enzymically with NADH (i.e., with coenzyme that contains only light hydrogen), the other enantiomer of monodeuteroethanol is formed (ea 4). The cvcle was comnleted when the cmfigurati~~n grf the chiral rnonodeuteroethanol was inverted. The alruhol was converted to its wsvlate. and the m s v late displaced by hydroxide ion (eqs 3 and 4): The resulting monodeuteuroethanol was then enzvmicallv oxidized bv NAD u,ith quantitati5.e transfer of de"terium-from the alcihol to the cnenryrne. The two hvdnraen atoms of ethanol are described toda; as enantiotopfc a n i the stereochemistry of methylene groups with enantiotopic hydrogen atoms is well understood. The idea should really have been well understood in 1951, since Ogstonin 1948had discussed the fundamental concept in connection with the enantiotopic carhoxymethyl groups of citric acid (13). Nevertheless, the enzymic reactions cited above did much to familiarize chemists and biochemists with the idea. In the most recent edition of Morrison and Boyd, the enzymic oxidation of alcohol is used to introduce the concept of enantiotopic and diastereotopic atoms (14). . . Here is an example wheie an important concept in stereochemistry was introduced into phvsical oreanic chemistrv from biochemistry.
The next coenzyme for which a mechanism was estahlished was thiamine pyrophosphate. Ronald Breslow, in 1957, used NMR spectroscopy to show that the hydrogen atom a t C-2 in a thiazolium salt rapidly exchanges with deuterium from DzO, even in only slightly alkaline solution (15). The exchange suggests that the coenzyme functions by offering an anionic center for catalysis. On the basis of this idea, Breslow put forward a mechanism for the action of thiamine pyrophosphate in the enzymic formation of acetoin. This mechanism (Fig. 2) parallels the mechanism that Lapworth had suggested (16) in 1903 for the benzoin condensation of henzaldehyde. Breslow (17) used this idea to elucidate the way in which thiamine functions in all of its reactions in- ~coou&ation -.-. ~ ~ . - ~ - with various enzvmes. Much suhsequent research has completely verified his interpretation. This is an earlvexamnle of the annlication of NMR svectros.. copy, one of the major tools of modern physical organic chemistry, to bio-organic chemistry. ~
~
~
.~~ ~~
~
Enzyme Mechanisms At the same time that the mechanisms of action of these three coenzymes were elucidated, the chemistry of four types of enzymes that function without coenzymes was worked out. All of this work occurred around 1950, but the carhest concerned the nction of sucrose phosphorylase, wh~chratalszrs the reversible reaction3 nfaucrose with inorganic phosphate to yield fructose and glucose-1-phosphate. In 1947, Doudoroff, Barker, and Hassid (18) postulated that the reaction involves the formation of an intermediate hetween glucose and the enzyme. Using radioactive phosphorous, they demonstrated that inorganic phosphate exchanges with glucose-1-phosphate in the presence of enzyme. In 1952, Fitting and Doudoroff (19) showed that fructose labelled with carbon-14 exchanges with sucrose under the influence of the enzyme,
Sucrose + enzyme-
i fructose
glucose-enzyme
-
Figure 2. Formation of acetoin,
I Enz I
BH
0
II I YH-CH,--0-P(OR),
Add
Hydrolysis
f phosphate
glucose-
1-phosphate + enzyme (5) Then, in 1953, Koshland (20) noted that this reaction occurs with retention of configuration a t C-l of glucose, and applied to this and to many other enzymic reactions one of the stereochemical principles of physical organic chemistry. He pointed out that a single displacement in enzymology, as in nonenzvmic chemistr;. should result in an inversion of con-~~~~~ figuration, whereas two displacements will lead to retention. The retention of confieuration observed with sucrose ~ h o s o " horylase was, therefore, consistent with the two-step mechanism offered hv Doudoroff and his co-workers. where a covalent intermediate between glucose and the enzyme was postulated. This stereochemical criterion has now been applied widely in enzymology, as it has in other chemical processes. Subsequently, the covalent intermediate between glucose and the enzyme has actually been observed. Almost simultaneously with the work on sugar phosphorylase, two separate groups developed the two-step mechanism for hydrolyses catalyzed by peptidases and esterases. Their mechanism. which nostulates an acvlated enzvme as intermediate, is the one that we accept today. The history of the mechanism of the serine esterases hegins, insofar as any scientific investigation can he said to have a precise beginning, with the discovery of the nerve gases during World War 11.In 1932, Lange and Kruger (21), in Germany, synthesized diethyl fluorophosphate and similar compounds and noted their general physiological effects, including their effects on eyes. They did not report a minimum lethal dose or any other quantitative measures of toxicity, but no one reading their paper would have missed the fact that the compounds are violently poisonous. During World War 11, Adrian and his co-workers (22) noted the similarity between the action of nerve gases such as the
.
~~
0 ,
Figure 3. Dialkyl fluorophosphateattack on acetylcholine esterase.
fluornphosphatea and that of reversible inhibitors of choline eswrase. This led ro a number of scientific investiaationj of the action of nerve gases on various esterases. Figure 3 shows the chemistry of the attack of a dialkyl fluorophosphate on the serine residue of one of the enzymes, suchas acetylcholine esterase. In 1949, Jansen and Balls (23) and their collaborators demonstrated that d i i s . o ~ r.. o ~fluoronhos~hate vl reacts stoichiometrically with chymotrypsin, a n i they crystallized the resultiue diisonro~vl~hos~horvlchvmotrwsin. ~ a c h m a n s o h n a n d~ i l &'(24)'and-their gro;p found other active . nhosnhorvlatine aeents. includhe tetraethvl. . pyrophosphate, that also re& ~toichiometrica& with ~ c e tylochuline esterase to inhibit it irrcversil)ly. In 1950, they and t'. Hergman (2.5)puhhihed the two-step mechanism fur the action ofacetvlcholine esterase that is shown in Figure 4. They marked the reactive group on the enzyme as " G , and drew a formula with a rather strange double bond from substrate to enzyme. Despite this double hond, they presented the essential idea of a two-step mechanism, where the acvlation of the enzvme is followed hv the hvdrolvtic cleav" " age of the acyl enzyme. In 1952. Hartlev and Kilbv (26) a t Camhridee allowed D nitrophenilacetaie to react"with'chyrnotryp&n and found that the kinetics showed a "burst". The curves for the production of p-nitrophenol do not extrapolate smoothly td the origin, hut instead are consistent with a verv rapid, initial reaction followed by a slower hydrolysis where the rate is proportional to the concentration of the enzyme. Furthermore, the amount of p-nitrophenol that is formed almost instantaneously from enzyme and p-nitrophenylacetate, i.e., Volume 63 Number 5 May 1986
411
0 G-H
+
1 I
R'-C-OR
washed out of the carbonyl group by other reactions. This work provided a basis for understanding an enzymic pathway involving a ketimine.
==+
II
CH,-C-CH,-COP
H-+
412
I
+ co,
CHrC=CH,
+ R-C-OHII
the "burst", corresponds to one mole of p-nitrophenol for each mole of chym&rypsin present in solution. ~ m t l e yand Kilby concluded that the hydrolysis proceeds by the very rnnid acetvlation of the enzvmeat itsactive site. followed bv thk slowe; hydrolysis of tge resulting acetylchymotrypsi~, regenerating the enzyme for further rapid reaction with the suhstrate. I n subsequent years, much evidence has been adduced to support this mechanism. In 1953, Balls and Wood (27) isolated serine phosphate from the hydrolysis of diisopropylphosphoryl chymotrypsin. Suhsequent work by Koshland and Kennedy (28) has shown that the enzyme is directly phosphorylated on serine and that the initial compound is not an artifact. Although p-nitrophenyl acetate is not a natural substrate for chymotrypsin, subsequent work has shown that normal substrates react in the same way that i t does. The confusion that surrounded the kinetics of the enzymic hydrolysis of estersand amides was resolved in 1962 by Bender and Zerner (29) and the X-ray structures of the hydrolytic enzymes are in accord with the deductions from chemistw. The mechanisms m t u l a t e d in the early '50's have been expanded and modified hut, in their ess&tials, they have been confirmed. A third early mechanism for enzymic processes involved standard imine chemistry. In 1959, Gordon Hamilton and I (30) postulated a ketimine mechanism for the decarhoxylation of acetoacetic acid. The mechanism was based on the physical organic chemistry of Kai Pedersen (31), who had previously postulated a rational sequence for the aniline catalyzed decarhoxylatiou of dimethylacetoacetic acid. The mechanism for the uncatalyzed decarboxylation of 8-keto acids had been established previously by Bredt (32) and by Pedersen. The acid loses carbon dioxide to form the enol of the product, which subsequently ketonizes. The idea behind Pedersen's mechanism for the aniline catalysis of decarhoxylation is that the amine reacts with the ketoacid to form a ketimine. Since nitrogen is more basic than oxygen, the ketimine can be ~ r o t o n a t e dmore readilv than the ketone, and the protonaied imine then provides a better electron sink than does the carhonyl group of the ketone. Although Pedersen offered little or no experimental support for his hv~othesis,it ~ r o v i d e da physical organic foundation for the riichanism o f t h e corresponding enzymic process. Hamilton labelled the carbonyl group of acetoacetic acid with ' 8 0 and then carried out the enzymic decarboxylation; he was able to show that the product of the decarhoxylation, acetone. contained This result is demand~ ~ ~ ~ ~ none ~ - of-the - lahel. , ed by the ketimine mechanism, whereas the mechanism of the uncatalvzed decarboxvlation would have reauired that the label appear intact in ihe product. o f course,;n order to make these statements. Hamilton had to carw out an elaborate set of control exp&nents t o show that the label is not ~~
H\,/R
i fw ti% m a c t i i of acetylcholine esterase.
Figure 4. Two-step ~
~
-
This type of mechanism was promptly confirmed, for the decarboxylation and for the physioloeically much more important reaction of transaldolase sudied by Bernard Horecker und his collaborators (331.The irnine intermediates in both cases were trapped by a method introduced in 1958 by Edmond Fischer and E. G. Krebs (34). They discovered that the ketimine linkaee - hv. which Dvridoxal Dhosohate is attached to phosphorylase A can be"reducedby sodium borohydride without inactivating, and hence without seriously damaging, the enzyme. In 1961, Horecker and his co-workers used horohvdride to reduce a mixture of transaldolase and glucose-6-phosphate labeled with 14C. In this case the process inactivated the enzyme, showine that a ketimine had heen formed a t the active site. On hy&olysis, the reduction product yielded a compound that contained '4C, and presumably involved the elements of D-erythrose attached to lysine. Suhsequent work by Horecker and his co-workers showed that aldolase, like transaldolase, is a ketimine enzyme (35). In 1962, Fridovich (36) showed that the addition of sodium horohvdride to a mixture of acetoacetate decarboxylase and acetoacetate inactivated the enzyme, whereas the addition of borohvdride to a buffer aolution of the enzyme alone had no effect on its activity. Hydrolysis of the reaction product yielded c-isopropyllysine formed by the reduction of the ketimine of acetone. Control experiments showed that this ketimine is actually an intermediate in the enzymic pathway. The mechanism for the action of rihonuclease was also sorted out in the early '60's and should properly he included in this exposition. Further, this discussion concerns only ~ a t h w a vfor s catalvsis. Nothine has been said concernine the reasons why enzymes cause enormous increases in rates. Nothine has been said about the concent of an e n t r o w t r a ~ . or the concept of approximation (that fs to say, bringing t& reactants close together in space), or the effect of changing the local polarity of the solvent by holding the substrates for an enzyme in a cleft in the rotei in. All these ideas, too, derived from physical organic-chemistry and have a proper place in this history, but onlssomuch can he crammed intoa short paper. I hope I have presented what happened or, a t least, some of what happened in the critical years from 1947 to 1963 when chemists placed the mechanisms of enzyme action on the firm footing of physical organic chemistry.
~~
Journal of Chemlcal Education
Literature Cited (1) Hammett. L. F."PhyaidOlganie Chsmiatry": MeCrsw-Hill:Now York, 1940.
(2) Sumner, J. B. J.Biol. Chm. 1926,69,435:Northrop,J. H. etal. J. Con.Phyaiol.1930,
13,739; 1932,16.267; 1935,18,433. (31 Sanger, F.; Tuppy, H. Biorhm. J. 1951,49,463,4m; Sanger, F.:Thompson,E. 0. P. Biorhrm. J. 1953,53,353.368.
(201 Koshlsnd. D. E.,Jr. B i d Reu. 1353.28.416. (211 Lenge. W.:Krueger,G. Ber. 1932, (22) Adrian. E. D.: Feldberg, W.; Kilby
2,859. (9) Brsun8tein.A. E.:Shemyakin, M.M. Biokhimiyo 1953.18,393. (lo) Mandelea, S.;Koppelman,R.;Hanke, M, E. J . B i d . Chem. 1954,209,327. (11) Snell.E.E.ets1. J.Amer.Chem.Soc. 1945.67,19+ 1952,74,919:1954,76,637,653:J. R i d . Chem 1352,199,699. (12) Westheimer, F. H.; Fisher, M. F.: Cann E. E.: vennesland, B. J. Amen Chem. Sac. lSdQ 7. 5. . ...,71 . .,.
(13) Ogston, A. G.Nature 1948,162,963. (14) Morrison, R.:Bqvd,R:~OrganicChemistry".
""--.". CA"
4th ed.;Allyn &Bacon: Boston.1983:pp
7c
(15) Bmslow, R J A m a r . Cham.Soc. 1957,79,1762. (161 Lsgworth,A. J . Chem.Soc. 1803,83,995. (17) Brsdow,R. J.Amor. Chem.Soc. 1958,80,3719. (18) Doudoroff.M.;Barker. H.A.; Hassid. W. 2.J.Bioi. Chem. 1947,168,125. (19) Fitting, C.: Doudoroff, M. J . B i d Chem. 1952,199,153.
"""". (35) Pontremo1i.S.; Prandini, B. D.: Bonsignore, A,: H0mker.B. L.Proc.Nof. Arod. Sci.. USA 1961.47,1942. (361 Fridovich. I.; Westheimer, F. H. J . Amer. Chem. Soc. 1962.84.3208.
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Number 5
May 1986
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