An Enzyme-Catalyzed Resolution Amino Acids
Jerry R. Mohrig and Samuel M. Shapiro Carleton College Northfield, Minnesota 55057
Although organic chemists have shown an increasing interest in the study of enzymic catalysis, enzymes are rarely found in an organic chemistry laboratory course. They have the reputation bf being too unstable and too complex. However, considerable progress has been made toward understanding the mechanisms by which enzymes catalyze chemical reactions (1,2).It is now possible to ask questions concerning the detailed molecular events of many enzymic catalyses with some hope of finding answers. The experiment that we wish to report uses papain, area.sonably stable hydrolytic enzyme, as a catalyst for the stereospecific formation of an insoluble anilide from a racemic henzoylamino acid. I t is designed for the undergraduate organic chemistry laboratory (3)and has been used a t Carleton for two years. The experiment combines classical, synthetic organic chemistry, a simple effective method of optical resolution, and the consideration of how papain catalyzes the hydrolysis of amides and esters. The experiment is rugged, reliable, and inexpensive. Those who like to relate their experiments to everyday living can substitute a grocery-store meat tenderizer, which has papain as its active ingredient, as the catalvst for the resolution. The experiment involves two separate steps. The fmt is the svnthesis of a henzovlamino acid from a racemic a-amino acid i d henzoyl chloride. 0
11
--
+ + H,N-CH-C0,-
C6Hj-C-CI
NsOH
I
C,H,--C-
II
HCI
eenerallv eives a oroduct that is satisfactory. for oolarimetric . &easur&ents. The specific rotations are large enough to make estimations of optical purity quite feasible. Papaln The enzyme papain (5) is a protein composed of a single polypeptide chain with a molecular weight of about 23,000. Its complete structure and stable conformation have been determined hv amino-acid analvsis and X-rav diffraction studies (6). Papain is found in the milky juice of the papaya fruit. Corica .DoDn>o . . Althoueh it constitutes 5%of the sduhle latex protein from the its hidogical role there is still amhieuous. The term ~ a o a i nis used for rhe crude dried lawx as weil as for the crys&line enzyme. We have used an extract of crude papain, since it is inexpensive, yet effective in the asymmetric synthesis. Papain is remarkably stable in the presence of organic solvents and to changes of pH, temperature, and ionic strength. I t is stable enough in the commercial crude powder to store at 25°C for a few days, although for prolonged periods it should be kept at 0-5OC. The digestant action of papaya juice has been known and used for centuries. The first controlled ex~erimentswere published just over 100 years ago and many investigations on naoain have been carried out durine the last centurv. . P a.~ a i n is known to catalyze a number of acyl addition-elimination reactions. Most important is the hydrolysis of amide bonds, although it is also an effective catalyst for the hydrolysis of a variety of esters and thiol esters. In fact, a common standard for the measurement of its catalytic activity is the ethyl ester of a-N-benzovl-L-areinine. Paoain cleaves a wide varietv of " peptide bonds; in dilute aqueous solution the equilibrium position strongly favors the hydrolysis products.
..
NH-CH-C02H
I
R This standard experiment has been used most commonly with glycine as the amino acid ( 4 ) . However, substitution of other amino acids works just as well, as long as quantities of reactants are changed so that their molar ratios are constant. We have successfully used leucine, valine, and alanine. The second step of the experiment is the papain-catalyzed formation of the L-anilide from the DL-benzoylamino acid 0
II
(DL>C,H,-C-NH-CH-C02
I
+ C6Hs-NH2
papain
R
R The anilide product is insoluble in the aqueous buffered reaction mixture and can easily he recovered by simple filtration. Purification by alkaline and water washes followed by drying 586 / Journal of Chemical Education
Studies on synthetic model compounds of low molecular weieht show that reaction at the carhonvl " . mouns - of L-areinine and L-lysine derivatives is fastest. The dissymmetric papain molecule has a hieh chiral snecificitv for the L-confieuration " of all substrates. Most oroteins are extensivelv" deeraded hv the action of papaiu. This explains its use as a meat tenderizer. A second commercial annlication is found in the brewine industw. Some .. brewers add small amounts of papain to prevent bottled beer from hecomine cloudv when chilled: the cloudiness results from the preGpitatioh of proteins at low temperatures. A varietv of medical aoolications have been r e ~ o r t e d(7).from use aisinst intestinai worms to the correction-of slippeddiscs. The importance of a sulfhydryl group to the catalytic action of papain has been recognized for some time ( 8 ) .First of all, the enzyme is inactivated by mild oxidizing agents, e.g., hromine, hydrogen peroxide, and oxygen. Activation reoccurs
.
n
-
when thiol compounds, such as cysteine, 2-merraptnethanol, or 2.3-dimerca~topropanol,are added. The active thiol group in unactivated-papain is thought to be present partly as a disulfide, partly as a sulfenic acid, and also in higher oxidation states. The addition of thiol compounds converts the disulfide and sulfenic acid forms to the mercaptan. Reducing agents, such as sodium horohydride, are also effective activators. Heavy metal ions, which complex sulfhydryl groups, are potent inactivators; EDTA and other chelating agents restore the activity. All of these, as well as other observations, are consistent with the importance of an enzymic thiol group in the catalytic action of papain. The irreversible inhibition of nanain hv, iodoacetamide results from the conversion of a single cysteine residue to the corresponding thioether through an h 2 reaction. Fxneriments with rarbon-14 laheled iodoacetale, X-ray structural studies, and other evidence indicate that the catalytically active thiol group of papain is on cyskine at position 25 in the sequence of 212 amino acid residues papain which make UD the . . molecule (5,6). There is general agreement that papain catalyzes the formation and hydrolyiii of amide and eiter hmds through the facile formation and suhsequcnt hydrolysis of an ncyl-enzbme intermediate. In the present ex~erimentthis thiol ester would have the following s&cture.
Enzvme-CH,SH.
0
. ~ ~ . ~ ~ ~~
~
II
R-C-NHR'
z2
Acyl enzyme
This mechanism is similar to the pathway by which achymotrypsin catalyzes the hydrolysis of amides and esters (101, with the exception that the catalytic site of that enzyme contains a serine hydroxyl group rather than a cysteine thiol. The intensively studied a-chymotrypsin system has also provided two excellent laboratory experiments, which have appeared in this Journal (11,IZ). Net formation of the L-henzoylamino acid anilide is favored by an excess of aniline and, more importantly, by the insoluhilitv of the anilide in the aaueous reaction mixture, which effeEtively removes it from the solution. So the equilibrium position which is usually far on the side of hydrolysis of amides and esters here strongly favors formation of the amide. The original observations of Bergmann and Fraenkel-Conrat (13) on the papain-catalyzed reaction of benzoylglycine and aniline provided some of the first critical experimental evidence for 'the enzymic synthesis of a peptide bind.' The; also realized the potential usefulness of papain for the optical resolution of a:amino acids. Although ;hi; asymmetric dynthesis has not proved to he a general method, it is effective when the solubilities of the reactant and product are in the right range and when the reaction stereospecificity is complete ( 1 4 ) .
~
In a few favorable cases the acyl papains are sufficiently stable to permit their direct examination by spectral and chemical methods (5). The minimal kinetic scheme for the hydrolysis of synthetic model compounds is a three-step mechanism (9). Either the acylation or deacylation step can he rate limiting, denendine u w n the structure of the earboxvlic acid derivative
Experimental Procedure 0
1I
+ R-C-NHR'
Enzyme-
Jerry R. Mohrig and Samuel M. Shapiro Carleton College Northfield, Minnesota 55057
Although organic chemists have shown an increasing interest in the study of enzymic catalysis, enzymes are rarely found in an organic chemistry laboratory course. They have the reputation bf being too unstable and too complex. However, considerable progress has been made toward understanding the mechanisms by which enzymes catalyze chemical reactions (1,2).It is now possible to ask questions concerning the detailed molecular events of many enzymic catalyses with some hope of finding answers. The experiment that we wish to report uses papain, area.sonably stable hydrolytic enzyme, as a catalyst for the stereospecific formation of an insoluble anilide from a racemic henzoylamino acid. I t is designed for the undergraduate organic chemistry laboratory (3)and has been used a t Carleton for two years. The experiment combines classical, synthetic organic chemistry, a simple effective method of optical resolution, and the consideration of how papain catalyzes the hydrolysis of amides and esters. The experiment is rugged, reliable, and inexpensive. Those who like to relate their experiments to everyday living can substitute a grocery-store meat tenderizer, which has papain as its active ingredient, as the catalvst for the resolution. The experiment involves two separate steps. The fmt is the svnthesis of a henzovlamino acid from a racemic a-amino acid i d henzoyl chloride. 0
11
--
+ + H,N-CH-C0,-
C6Hj-C-CI
NsOH
I
C,H,--C-
II
HCI
eenerallv eives a oroduct that is satisfactory. for oolarimetric . &easur&ents. The specific rotations are large enough to make estimations of optical purity quite feasible. Papaln The enzyme papain (5) is a protein composed of a single polypeptide chain with a molecular weight of about 23,000. Its complete structure and stable conformation have been determined hv amino-acid analvsis and X-rav diffraction studies (6). Papain is found in the milky juice of the papaya fruit. Corica .DoDn>o . . Althoueh it constitutes 5%of the sduhle latex protein from the its hidogical role there is still amhieuous. The term ~ a o a i nis used for rhe crude dried lawx as weil as for the crys&line enzyme. We have used an extract of crude papain, since it is inexpensive, yet effective in the asymmetric synthesis. Papain is remarkably stable in the presence of organic solvents and to changes of pH, temperature, and ionic strength. I t is stable enough in the commercial crude powder to store at 25°C for a few days, although for prolonged periods it should be kept at 0-5OC. The digestant action of papaya juice has been known and used for centuries. The first controlled ex~erimentswere published just over 100 years ago and many investigations on naoain have been carried out durine the last centurv. . P a.~ a i n is known to catalyze a number of acyl addition-elimination reactions. Most important is the hydrolysis of amide bonds, although it is also an effective catalyst for the hydrolysis of a variety of esters and thiol esters. In fact, a common standard for the measurement of its catalytic activity is the ethyl ester of a-N-benzovl-L-areinine. Paoain cleaves a wide varietv of " peptide bonds; in dilute aqueous solution the equilibrium position strongly favors the hydrolysis products.
..
NH-CH-C02H
I
R This standard experiment has been used most commonly with glycine as the amino acid ( 4 ) . However, substitution of other amino acids works just as well, as long as quantities of reactants are changed so that their molar ratios are constant. We have successfully used leucine, valine, and alanine. The second step of the experiment is the papain-catalyzed formation of the L-anilide from the DL-benzoylamino acid 0
II
(DL>C,H,-C-NH-CH-C02
I
+ C6Hs-NH2
papain
R
R The anilide product is insoluble in the aqueous buffered reaction mixture and can easily he recovered by simple filtration. Purification by alkaline and water washes followed by drying 586 / Journal of Chemical Education
Studies on synthetic model compounds of low molecular weieht show that reaction at the carhonvl " . mouns - of L-areinine and L-lysine derivatives is fastest. The dissymmetric papain molecule has a hieh chiral snecificitv for the L-confieuration " of all substrates. Most oroteins are extensivelv" deeraded hv the action of papaiu. This explains its use as a meat tenderizer. A second commercial annlication is found in the brewine industw. Some .. brewers add small amounts of papain to prevent bottled beer from hecomine cloudv when chilled: the cloudiness results from the preGpitatioh of proteins at low temperatures. A varietv of medical aoolications have been r e ~ o r t e d(7).from use aisinst intestinai worms to the correction-of slippeddiscs. The importance of a sulfhydryl group to the catalytic action of papain has been recognized for some time ( 8 ) .First of all, the enzyme is inactivated by mild oxidizing agents, e.g., hromine, hydrogen peroxide, and oxygen. Activation reoccurs
.
n
-
when thiol compounds, such as cysteine, 2-merraptnethanol, or 2.3-dimerca~topropanol,are added. The active thiol group in unactivated-papain is thought to be present partly as a disulfide, partly as a sulfenic acid, and also in higher oxidation states. The addition of thiol compounds converts the disulfide and sulfenic acid forms to the mercaptan. Reducing agents, such as sodium horohydride, are also effective activators. Heavy metal ions, which complex sulfhydryl groups, are potent inactivators; EDTA and other chelating agents restore the activity. All of these, as well as other observations, are consistent with the importance of an enzymic thiol group in the catalytic action of papain. The irreversible inhibition of nanain hv, iodoacetamide results from the conversion of a single cysteine residue to the corresponding thioether through an h 2 reaction. Fxneriments with rarbon-14 laheled iodoacetale, X-ray structural studies, and other evidence indicate that the catalytically active thiol group of papain is on cyskine at position 25 in the sequence of 212 amino acid residues papain which make UD the . . molecule (5,6). There is general agreement that papain catalyzes the formation and hydrolyiii of amide and eiter hmds through the facile formation and suhsequcnt hydrolysis of an ncyl-enzbme intermediate. In the present ex~erimentthis thiol ester would have the following s&cture.
Enzvme-CH,SH.
0
. ~ ~ . ~ ~ ~~
~
II
R-C-NHR'
z2
Acyl enzyme
This mechanism is similar to the pathway by which achymotrypsin catalyzes the hydrolysis of amides and esters (101, with the exception that the catalytic site of that enzyme contains a serine hydroxyl group rather than a cysteine thiol. The intensively studied a-chymotrypsin system has also provided two excellent laboratory experiments, which have appeared in this Journal (11,IZ). Net formation of the L-henzoylamino acid anilide is favored by an excess of aniline and, more importantly, by the insoluhilitv of the anilide in the aaueous reaction mixture, which effeEtively removes it from the solution. So the equilibrium position which is usually far on the side of hydrolysis of amides and esters here strongly favors formation of the amide. The original observations of Bergmann and Fraenkel-Conrat (13) on the papain-catalyzed reaction of benzoylglycine and aniline provided some of the first critical experimental evidence for 'the enzymic synthesis of a peptide bind.' The; also realized the potential usefulness of papain for the optical resolution of a:amino acids. Although ;hi; asymmetric dynthesis has not proved to he a general method, it is effective when the solubilities of the reactant and product are in the right range and when the reaction stereospecificity is complete ( 1 4 ) .
~
In a few favorable cases the acyl papains are sufficiently stable to permit their direct examination by spectral and chemical methods (5). The minimal kinetic scheme for the hydrolysis of synthetic model compounds is a three-step mechanism (9). Either the acylation or deacylation step can he rate limiting, denendine u w n the structure of the earboxvlic acid derivative
Experimental Procedure 0
1I
+ R-C-NHR'
Enzyme-
