COMMUNICATIONS TO THE EDITOR
July 20, 19.58
result of his extensive studies is now conclusively authenticated. RESEARCH LABORATORIES VETERANSADMINISTRATION HOSPITAL AND J. D. EDWARDS, JR. DEPARTMENT O F BIOCHEMISTRY BAYLOR UNIVERSITY COLLEGE OF MEDICINE HOUSTON, TEXAS RECEIVED JUNE24, 1958
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i
E 0
0
m
t
t v)
z
W 0
THE FORMATION OF a-IMINO ACIDS IN THE ENZYMATIC OXIDATION OF AMINO ACIDS‘ Sir :
J
Kinetic application of the borate-tautomerase system2 to the oxidation of L-tyrosine with ophio Lamino acid oxidase3 (Fig. 1)confirmed earlier demonstrations4Js6that theoxidase produced the keto tauto-
a u I-
a 0
I
2
3
5
4
6
MINUTES.
I
Fig. 2.-Oxidation of 1.2 pmoles of L-tyrosine in 3.2 ml. of 0.0375 M phosphate, 0.045 M KC1, pH 7.2, other conditions as in Fig. 1: curve 1, 8 mg. oxidase, no tautomerase; curve 2 , s m g oxidase, 0.025 ml. tautomerase; curve 3, 8 mg. oxidase, 0.2 ml. tautomerase; curve 4, 12 mg. oxidase 0.2 ml. tautomerase.
/
cc41* f
0 2
1
2
3
4
5 6 MINUTES
7
8
9
IO
Fig. 1.-Oxidation of 0.4 fimoles L-tyrosine in 3.5 ml. 0.57 M borate, pH 7.2, with ophio L-amino acid oxidase at 25’ : a, formation of non-absorbing keto-p-hydroxyphenylpyruvate. Addition of 0.2 ml. tautomerase a t 4 (A) or a t zero time ( 0 ) catalyzed equilibration with the strongly absorbing enol borate. All experiments contained 1 mg. catalase and 4 mg. crude venom of Agkistrodon piscivorus piscivorus.
mer of the a-keto acid. However, in phosphate or tris-(hydroxymethy1)-aminomethanebuffers tautomerase addition resulted in formation of a transient absorbing intermediate whose accumulation depended both on the tautomerase and oxidase concentrations (Fig. 2, curves 2, 3, 4). Without tautomerase a smooth rise in optical density from formation of the weakly absorbing keto-enol equilibrium mixture was observed (Fig. 2, curve 1). Tautomerase does not alter the keto-enol equilibrium and could produce only a steeper rise in optical density to the final equilibrium value. These results represent the first direct indication of Knoop’s’ imino acid intermediate in the oxida(1) This investigation was supported by Atomic Energy Commission Contract KO.AT(30-1)-901. ( 2 ) W. E. Knox and B. M. Pitt, J . Bioi. Chem.. 226, 675 (1957). (3) Obtained from Ross Allen’s Reptile Iustitute, Silver Springs, Fla. (4) A. Meister, Nature, 168, 119 (1951). ( 5 ) W.A. Fones. Arch. Biochem. Bicphys., 36,486 (1952). (6) C . Frieden and S. F. Velick, Biochem. Biophys. Acta, 23, 439 (1957). (7) F. Knoop, Z . physiol. Chem., 67, 482 (1910); F. Knoop and H. Oesterlin, ibid., 148, 294 (1925).
tion of amino acids. The humps in Fig. 2 result from the formation of the enamine tautomer of the a-imino acid, and represent up to 5% of the initial tyrosine concentration estimated from the extinction coefficients of enol p-hydroxyphenylpyruvate and a-N-acetylamino-9-hydroxycinnamate.I n the presence of tautomerase the reaction takes the course : RCHzCHCOOH
1
NHz
+ RCHzCCOOH
----t
II
RCHzCCOOH
NH
II
0
ti
tautomerase
RCH=CCOOH
I
NHz
It is assumed that tautomerase, which catalyzes the keto-enol tautomerization of the a-keto acids, also catalyzes t h e imine-enamine tautomerization of the a-imino acids. The relatively slow spontaneous rate of the latter tautomerization and the rapid imino acid hydrolysis preclude observation of this effect without tautomerase. Similar enamine accumulations were observed during oxidation of L-phenylalanine and L-tryptophan. The accumulation of the tryptophan intermediate required the presence of a new rat liver indolylpyruvate tautomerase and did not occur with pig kidney tautomerase which is inactive with indolylpyruvate.2 The interpretation of the humping effect as a transient enamine accumulation is thus supported by the specificity of indolylpyruvate tautomerase. It should be emphasized that the keto tautomer of the a-keto acid is the ultimate product of the L-amino acid oxidase reaction. Added tautomerase is essential for formation of the enamine, and from the formation of this intermediate i t follows that the imine tautomer is the initial amino acid
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COMMUNICATIONS TO THE EDITOR
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approximately twice those of Ascnrzs inuscle glycogen. The two conventional methods used for the (8) I*. I~ASS.4cIIL?SETTS ranging between 0 and 20'. The nioleculx weight RECEIVED APRIL 3,1Qt5X of w.;lter extrxted glycogen was found to be at least 10 times that of glycogen extracted with cold trichloroacetic acid and 50 to 100 times that of thc SEDIMENTATION CHARACTERISTICS OF alkali extracted material. FurtherrnorL heciting GLYCOGEN' above 00' resulted in a progressive degradation ol SI Y: water extracted glycogen. Glycogens which exhibit extremely high molecBecause of the observed high molecular wcights ular weight have been obtained by extraction of the possibility of aggregation of glycogen nioleculesi this polysaccharide with water a t low temperatures w'is tested using procedures known to disrupt elec(0 to 4') from Ascaris muscle and from rabbit trostatic bonds. Samples treated with urea (4 1/. liver. O",R one ~ c e k ) guanidine , hydrochloride ( 7 l/, 0')' Glycogen readily sedimented on ultracentrifuga- two days) or with anionic, non-ionic or cationic tion (50,000 to 60,000 X g; 0 to 4"). Under these detergents (sodium tloclecyl sulf,Ltc, 'rwecti VI, conditions over 957c of the total liver glycogen cetyltriinetli\-laiiiiiiotiiuiii bromide) ( I ( i, 20", 1 i was recovered in the residue. Protein and other hours) showed t i 0 chmqe dctectable by scdiriientc~impurities were removed by differential centrifuga- tion analysis. .'ilso, no changes were found oil tion, repeated mechanical shaking with a mixture repeated freezing and thawing, on repeated preof chloroform and octyl alcohol2and treatment with cipitation, drying and rediss )lving ant1 after macrotrypsin and chymotrypsin. The analyses of puri- bic incubation in 0.01 S KOH ( X " ,24 hours) In fied samples (dried in vacuo a t 75' to constant addition, aliquots taken :it various stages of the weight) which contained no detectable protein purification 1,rocedurc m c l coiitaining yogressix el! (less than 0.005'%) yielded the theoretical values lower concentrations of contdminating protciii for glycogen (Calcd. for (C6H1005)1L:C, 34.44; (from IC; to less than 0 003 ) showcd itleiitical H, 6.22; 0, 49.34. Found: C, 44.59; H, (3.59; Sedimentation characteristics, ndicatirig that thc 0,40.47). presence or absence of protein bears no relatiorishii) Ultracentrifugal analyses indicated weight aver- to the molecular si7e of glycogen. 011the b m s 0' age sedimentation coefficients of 300 to 1000 these observations the hrpothesls that the high svedberg units and corresponding minimal weight molecular weight of water evtracted glycoge~iis thc average molecular weights of 50 to 200 million. result of aggregation receive., no support. In the absence of reliable diffusion measurements Measurements of diffusion rates are in proyress the molecular weight is stated as the minimum and should permit accurate estitnates o f iiiolcc~il~ir possible for a material having this sedimentation weight. rate. DEP-4RlRIEYT O F PIIAR\L4C(II 0C.I Sctlinientation coefficient distributions showed SCHOOL OF MEDICIVE Siaxr E Y A ORRLII , T R extreme polydispersity and marked skewness, a LOUISIAYA Si ATE V \ n r R ~r \I ERWI-ST Bucnr\c. typical sample including small amounts below 25 S E I 1 ORLEAYS, I,OLIS1.4\.i R E C F I ~ GM-lr D 79, 1958 svedbergs and significant amounts above 2000 svedbergs, with the weight average near SO0 svedbergs. Glycogens extracted from both rabbit liver and Ascaris muscle yielded sedimentation distributions of similar shape. However, the sedimentatioil coefficients for liver glycogen were oxidation product. This refutes the suggestions that the enamine is the normal interme~liate.8.~
( 1 ) Sul,yorted b y grants ( E 4 0 8 and 2E-10) from the hTcitional I n.titutes of Health. United States Public Health Service ( 2 ) hr Rtcl
