Oxidative deamination of sulfur amino acids by bacterial and snake venom l-amino acid oxidase

ARCHIVES

OF BIOCfIEMISTRY

AND BIOPHYSICS

148, 5~-~i3 (1971)

Oxidative Deamination of Sulfur Amino Acids by Bacterial and Snake Venom k-Amino Acid Oxidase ~ S U S A N S. C H E N ,

JEAN

HUDSPETH

WALGATE,

AND J O H N

A. D U E R R E

Ireland Research Laboratory, Department of Microbiology, School of Medicine, University of North Dakota, Grand Forks, North Dakota 58201 Received January 18, 1971 ; accepted May 24, 1971 We have investigated the oxidative deamination for all available sulfur amino acids by the erystMline snake venom L-amino acid oxidase from Crotalus terrificus terrificus and a particulate bound oxidase from an atypical Proleus rettgeri. Specific activity, pH optimuni, K,, and V ..... values were determined for each sulfur amino acid. For the bacterial enzyme the affinity for the various substrates at pH 7.5 iti decreasing order is L-honloeysteine, L-eysteine, L-methionine, L-honmeystine, S-ribosylL-homocysteine, S-adenosyl-L-homoeysteine, S-adenosyl-L-methionine, methionine sulfoxide, L-djenkolic acid, S-adenosyl-L-homoeysteine sulfoxide, and L-eystine. No measurable activity was observed with cysteic acid, homocysteie acid, lanthionine, eystathionine, or S-ribosyl-L-homoeysteine sulfoxide. The snake venom enzyme had a somewhat more limited specificity. There was no measurable activity with any of the sulfoxides, S-adenosyl-L-methionine, eysteic acid, homocysteic acid, lanthionine, or eystathionine. The affinity for the various substrates at pH 7.5 in decreasing order is L-methionine, L-homoevstine, L-homoeysteine, S-adenosyl-L-homoeysteine, S-ribosyl-L-homoeysteine, L-djenkolic a d d , L-eysteine, and L-eystine. The keto acids produced from the various substrates with both enzymes were identified by chemical analysis and by thin-layer chromatography of the free keto acid or their dinitrophenylhydrazones against known standards. All the resultant keto acids appear to correspond to their parent compounds. Homocystine was found to be completely oxidized to 4,4'-dithio-bis (2-ketobutyrie acid). S-Adenosyl-Lmethionine, which is resistant to attack by most enzymes including snake venom L-amino acid oxidase, was readily oxidized to S-adenosyl-a-keto-v-methiolbutyrate by the bacterial enzyme. The mercapto-a-keto acids derived from homoeysteine and cysteine were found to be unstable resulting in the liberation of hydrogen sulfide and c~-ketobutyrate or pyruvate, respectively. L-Amino acid oxidases h a v e b e e n isol a t e d from s e v e r a l sources i n c l u d i n g microo r g a n i s m s (1 3), s n a k e v e n o m (4, 5), r a t k i d n e y (6, 7), b i r d livers (8, 9), a n d i n v e r t e b r a t e s (10, 11). T o d a t e a c t i v i t y of this enz y m e t o w a r d sulfur a m i n o acids h a s b e e n d e m o n s t r a t e d o n l y for the following sources: Neurospora crassa on cysteine, cystine, cystat h i o n i n e (12); Mytilus edulis on m e t h i o n i n e ,

e y s t a t h i o n i n e , d j e n k o l i e acid, e y s t i n e a n d h o m o e y s t i n e (10) a n d Crotalus adamanteus oil e t h i o n i n e , m e t h i o n i n e , h o m o c y s t i n e , a n d e y s t i n e (13). R e c e n t l y M i l l e r a n d D u e r r e (14) r e p o r t e d t h a t the r a t k i d n e y L-amino acid oxidase c a t a l y z e d t h e o x i d a t i v e d e a m i n a t i o n of S - a d e n o s y l - L - h o m o c y s t e i n e , S - a d e n o s y l L-methionine, and homocysteine. I n t h e p r e s e n t s t u d y b o t h the L-amino a c i d o x i d a s e s f r o m Proteus rettgeri a n d Crotalus terrificus terrificus v e n o m were q u i t e a c t i v e t o w a r d s e v e r a l sulfur a m i n o acids. T h e two e n z y m e s differed s o m e w h a t in s u b s t r a t e s p e c i f i c i t y a n d t h e r a t e s of a c t i v i t y .

i This investigation was supported by National Science Foundation Grant GB 7545 and by Public Health Service Research Developmevt Award 1-K3-GM-25, 962-05 to Dr. Duerre. 54

L-AMINO ACID OXIDASE EXPERIMENTAL PROCEDURES

55

t~moles phosphate buffer, pH 7.5, and 9.6 mg enzyme protein in a final volume of 3.0 ml. The particular fraction contained sufficient catalase L-Homocystine was obtained from Mann Re(6.1 ~moles H202/min/mg protein) to decompose search Laboratories, Inc., and L-eystine, L-homo- all hydrogen peroxide formed (21). After 30 rain cysteine-thiolactone-HC1, L-methionine, L-lan- of incubation at 30~ the re,'~ction mixture was dethionine, L-cysteine (free base), and L-methionine proteinized with 0.1 ml 100% trichloroacetic acid sulfoxide from Calbiochemicals. L-Cystathionine, and keto acid, H2S, NH~ were measured as outL-djenkolic acid, pyruvate, and a-ketobutyrate lined above. The pH optimum was determined by were obtained from Sigma Chemical Co. Bromo- the rate of oxygen consmnption using the phospyruvic acid was obtained from Eastman Organic phate buffer system. Chemical Co. L-Homucysteine was prepared from K~ deLerminations. K,,~ and V~,~ for all subL-homocysteine thiolactone with alkali (15). S- strates were determined from Lineweaver-Burk Adenosyl-b-homocysteine, S-ribosyl-L-homocys- double-reciprocal plots of initial reaction velociteine, and their solfoxides were prepared as out- ties of keto acid formation as a function of sublined previously (16). S-Adenosyl-L-methionine strate concentration. The rate of oxygen conwas prepared by the method of Schlenk, Dainko, sumption was determined polarographically using and DePalma (17). ~-Mercaptopyruvate was prea Gilson oxygraph. pared from bromopyruvate as described by K u a Chromatography. Ascending thin-layer chroma(is). tography was performed in glass jars on plates Enzymes. The crystalline L-amino acid oxidase coated with cellulose. Samples were applied in from Crotalus terrificus terrificus venom and the aqueous solution and two solvent systems, (1) bovine liver catalase were purchased from Boeh- ethanol-acetic acid-water (65:1:34) and (2) ringer, Mannheim. The particulate bacterial propanol-acetic acid-water (65:4: 31), were used. n-amino acid oxidase from Proteus rettgeri ~ was The method of Dancis, Hutzler, and Levitz for prepared by the method of KrishnamurtM, Buck- keto acid identification by thin-layer chromatogIcy, and Duerre (21). raphy was used (27). The hydrazine derivatives of keto acids were prepared by the addition of a 20% Methods molar excess of 0.01 M 2,4-dinitrophenylhydrazine Analytical procedures. Protein concentration in 2.0 N HC1 to the reaction mixture. After approxiwas measured by the method of Lowry et al. (22) ; mately 4 hr the crystals were collected by cenketo acid by the 2,4-dinitrophenylhydrazine trifugation and washed three times with cold water and dissolved in ethanol. Aliquots were method of Friedemann and Haugen (23); ammonia applied to thin-layer silica gel plates which had was obtained by diffusion using the method of been previously activated at 110~ for 1 hr. The Braganca, Quastel, and Schucher (24); and hysolvent systems used were isoamyl alcohol-0.25 N drogen sulfide was determined by the colorimetric ammonium hydroxide (20:1) and 4.0% ammonium method (25). Enzyme assays. The oxidation of the various hydroxide saturated with butanol. substrates was measured by determining the rate RESULTS of oxygen consumption manometrically (26). The vessels contained 15 t~moles of substrates, 300 T h e a b i l i t y of the p a r t i c u l a t e b o u n d bac~moles phosphate buffer, pH 7.5, 300 units beef terial L-amino acid oxidase to catalyze the liver catalase, 250 ~moles KC1, and 25 t~g crystal- oxidative d e a m i n ~ t i o n of sulfur amino acids line snake venom L-amino acid oxidase in a final was tested. N o m e a s u r a b l e a c t i v i t y was obvolume of 3.0 ml. For the bacterial enzyme the served with cysteic acid, homocysteic acid, vessels corltained 15 ~moles of substrate, 300 l a n t h i o n i n e , c y s t a t h i o n i n e , or S-ribosyl2 This organism was originally classified as an h o m o c y s t e i n e sulfoxide. T h e rate of o x y g e n Achromobacter species (19). However, due to its c o n s u m p t i o n a n d p r o d u c t f o r m a t i o n from limited activity on carbohydrates it has been re- those sulfur a m i n o acids oxidized are shown classified as an atypical Proteus rettgeri. In con- i n T a b l e I. trast to typical Proteus, this organism is nonT h e r a t e of p r o d u c t f o r m a t i o n versus oxymotile, has a lower optimal growth temperature (24~ produces hydrogen sulfide from cysteine, gen c o n s u m p t i o n followed the characterisand gives a negative test for indole on standard tic p a t t e r n o b s e r v e d for a general L-amino acid oxidase, i.e., the r a t e of oxygen conpeptone or tryptophan medium. However, the organism gives a positive indole test on nitrate s u m p t i o n to keto acid a n d a m m o n i a formed medium (20). was a p p r o x i m a t e l y 1 : 2 : 2 , except for the Materials

56

CHEN, WALGATE, AND DUERRE TABLE I

I~,ATES OF OXYGEN CONSUMPTION AND PRODUCT FORMATION FROM SULFUR AMINO ACIDS BY A BACTERIAL L-AMINO ACID OXIDASE a Specific actlvityb Substrate 02

Keto acid NH~

H2S

g~ S-Adenosyl-L-methionine S-Aden osyl-L-homocysteine S-Adenosyl-L-homocysteine sulfoxide S-I~ibosyl-L-homocysteine L-1V[ethionine L-Methionine sulfoxide L-Homocysteine L-Homocystine L-Cysteine L-Cystine L-Djenkolie acid

1.6 ~3.8 .)2.5 0 9.3 [9.7 17.4 0 0.5 1.1 1.0 0 4.0 3.3 0.9 8.8 0.0 3.3 1.2 2.4

8.4 ~6.5 1.8 5.8 ~2.0 2.4 3.8 4.5

7.91 0 ")4.11 0 1.81 0 6.01 0.3 .)4.61 0 2.4i 0.1 2.5 Trace 4.1 0 I

Oxygen consumption was measured manometrically. The vessels contained 15 #moles substrate, 300 #moles phosphate buffer, pH 7.5, and 9.6 mg enzyme protein in a final volume of 3.0 ml, After 30-rain incubation at 30 ~ the reaction mixtures were deproteinized w i t h T C A and keto acids

and t-I2S measured as outlined under Methods. Ammonia was measured in a duplicate vessel by the diffusion technique of Braganca et al. There was no oxygen consuInption or product formation in endogenous controls. Oxygen eonsumption remained linear throughout the experiment. b Activity is expressed as mtmmles of oxygen eonsumed or products produeed/min/mg protein. free sulfhydryl compounds, homoeysteine and cysteine. With these compounds the rate of oxygen consumption was greater than one. The excess oxygen consumption m a y be due to autooxidation of the parent sulfhydryl compound or of the products. Of interest was the observation t h a t H2S was liberated fl'om cysteine and homocysteiue (Table I). No H.~S was liberated under anaerobic conditions nor did pyridoxal phosphate affect the rate of formation of H2S, hence, it was unlikely t h a t the particulate fraction from the bacteria contained a desulfhydrase. When 5 - m e r c a p t o p y r u v a t e was utilized as substrate in the reaction, measurable amounts of HeS were formed. Since a corn-

parable amount of HaS was formed in the absence of enzyme, it was concluded t h a t the mercapto keto acids derived fl'om cysteine and homoeysteine were somewhat labile under the conditions employed. Product identification. Reaction mixtures incubated as outlined in Table I were deproteinized with TCA, filtered, and the residual T C A removed b y three extractions with an equal volume of ether. After chromatography on thin-layer cellulose plates keto acids were located with 2,4-dinitrophenylhydrszine. Quadruplicate sets of plates developed i~ ti~e same solvent system were sprayed with ninhydrin, platinic iodide, or ammoniacal silver nitrate (Table II). Tile keto acids derived from S-adenosylmethichine, S-adenosylhomocysteine, and Sribosylhomocysteine, all reacted with iodoplatinate and ammoniaeal silver nitrate indicating that the sulfur atom and earboh y d r a t e moiety remained associated with the keto acid derivative. In addition the keto acid derived from S-adenosylhomocysteine and S-adenosylmethionine absorbed ultraviolet light indicating the presence of the purine moiety. In the reaction using S-adenosylmethionine as substrate it was found that methylthioadenosine and homoserine were present. However, no activity was observed when L-homoserine was incubated with enzyme eliminating this compound as the active substrate. I n addition, the RF values and chromatographic properties of the keto acids derived from S-adenosyl-L-homocysteine and S-adenosylL-methionine were identical to those previously reported (14, 28). T h e end product from homocystine was studied quite intensively to determine if either one or b o t h the amino groups were eliminated. Results of chromatographs with reaction products from time course studies revealed t h a t mixed products resulted early in the reaction, whereas only one keto acid was observed after prolonged incubation (Table II). The 2,4-dinitrophenylhydrazones of early and late reaction products from homoeystine were also prepared. Chromatographic results again indicated t h a t more than one product resulted early in the reaction, whereas two

L-AMINO A C I D O X I D A S E TABLE II I { p VALUES OF K E T O ACIDS D E R I V E D FROM SULFUR AMINO ACIDS BY BACTERIAL L-AMINO ACID OXIDASE a

R~ Keto acids derived ~ronl

S-Adenosyl-L-methionine S-Adenosyl-L-hom~Vocysteine S-I~ibosyl-I~-homocysteine L-Methi(mine L-Djenkolic acid ~-Homocystine (1 hr) (4 hr)

Ethanol:acetic acid water (65:1:34)

Propanol: acetic acid:water (65:4:31)

0.76

0.77

0.62

0.55

0.79

0.69

0.81 0,30

0.73 0.10

0.39 0.60 0.60

0,41 0.53 0,55

/~-Mereaptopyru-

of 446 calculated for a monoaminomonoketo derivative. Results of chemical analysis are also in agreement with the presence of two keto groups in the parent compound. Therefore, the ultimate product, from homoeystine was confirmed to be 4,4'-dithio-bis, (2-keto butyric acid). COOH- I~-CH2- CH2-S-S-CH 2 -CH2-C-COOH " II N I NH I

N I NH I

C6H.~(NOz)z

e--~H3 (NOa)z

CzoHm Ola Ns S2 Calculated: C38.4

~-Homoeysteine (1 hr) (4 hr) a-Ketobutyrate (control) L-Cysteine (1 hr) (4 hr) P y r u v a t e (control)

57

0.13 0.36 0.880.21 0.46 0.82 0.39 0.89 0.21 0.44 0.85 0.88 0.84

0.46 0.76 0.44 0.81 0.78 0.48

0.45 0.65 0.46 0.66 0.66 0.46

r a t e (control) T h e experinIentM conditions were similar to those o u t l i n e d under 'Fable I except 30 #moles of s u b s t r a t e were used. After 1-hr incubation the reaction m i x t u r e s were deproteinized with T C A a n d c h r o m a t o g r a p h e d in q u a d r u p l i c a t e on t h i n - l a y e r cellulose plates. ResiduM s u b s t r a t e s were located on one set of plates w i t h n i n h y d r i n , keto acids on another with 2,4-dinitrophenylhydrazine, thioe t h e r and sulfoxide on a n o t h e r , w i t h i o d o p l a t i n a t e a n d c a r b o h y d r a t e on a f o u r t h with a m m o n i a e a l silver n i t r a t e .

isomers of a single product were indicative after 4-hr incubation (Table III). Sufficient quantities of the dinitrophenylhydrazone fl'om the 4-hr reaction mixture were prepared for chemical analysis. The melting point of the dinitrophenylhydrazone was 164~ The molecular weight as determined in acetone was 687, some 10% higher than 626 calculated using the formula for a diketo derivative. However, these results do not overlap with a molecular weight

Found

H 2.9 0 3Q6 N 17.9 S 10.3

C :39.4 H :3.7 0 29.2 N 15.2 S II .6

Sufficient quantities of the early reaction products from homocystine could not be prepared readily for analysis. However, the keto acid-positive compounds having the RF values of 0.39 in ethanol-acetic acidwater system also reacted with ninhydrin. This suggested that a mono-keto-aminoamino intermediate was formed. Results of chromatographic studies with homoeysteine as substrate were somewhat ambiguous. L-Homoeysteine tends to streak having an Rs of 0.66-0.71, and 0.43 in ethanol-acetic acid-water, and propanolacetic acid-water, respectively. Moreover, otber reaction products were also present, some of which appeared to have the same RF values and chemical properties as homocystine and homoeysteie acid. Since homocystine was present in the reaction mixture, the keto acid having an RF value of 0.36 in the ethanol-acetic acid-water system might be derived from this compound. In addition, two other keto acids were present. Results of chromatography of the dinitrophenylhydrazones of the keto acids derived fl'om the free sulfhydryl compounds were somewhat more meaningful. The major reaction products derived from cysteine after 1-hr incubation corresponded to the two isomers of ~-mercaptopyruvate, whereas after prolonged incubation two additional spots appeared which corresponded to the two isomers of pyruvate. We did not pre-

CI-IEN, WALGATE, AND DUERRE

58

TABLE I I I RF VALUES OF DINITROPHENYLtIYDRAZONES OF K E T O ACIDS D E R I V E D FROM SULFUR AMINO ACIDS BY BACTERIAL L-AMINO ACID OXIDASE a R~ Keto acid derived from

S-Adenosyl-L-methionine S-Adenosyl-L-homocysteine S-Ribosyl-L-homocysteine L-Methionine L-Djenkolic acid L-Homocystine (1 hr) (4 hr) L-Homocysteine (1 hr) (4 hr) a-Ketobutyrate (control) L-Cysteine (1 hr) (4 hr) Pyruvate (control) /3-Mercaptopyruvate (control)

Isoamyl alcohol:0.25 N NH4OH (20:1)

0.47 0.02 0.16 0.36 0.00 0.09 0.10 0.03 0.02 0.13 0.04 0.05 0.09 0.05

0.35 0.07 0.21 0.19 0.12 0.13 0.28 0.24 0.12 0.23 0.30

0.44 0.27 0.30 0.23 0.31

Butanol saturated with 4.0% NH4OH

0.81 0.21 0.59 0.60 0.22 0.06 0.19 0.26 0.24 0.47 0.23 0.22 0.38 0.23

0.27 0.17 0.27 0.34 0.34 0.64 0.35 0.33 0.54 0.33

0.26 0.64 0.45 0.68 0.48 0.65 0.37 0.56

a The experimental conditions were similar to those outlined under Table II. After 1-hr incubation the reaction mixtures were deproteinized with TCA and dinitrophenylhydrazone prepared as outlined under Methods. The resultant dinitrophenylhydrazones were dissolved in alcohol and chromatographed on silica gel. pare 7-mercapto-a-keto-butyrate due to technical difficulties; however, it would appear t h a t this compound is the resultant product from the oxidative deamination of homocysteine, which upon prolonged incubation underwent spontaneous decomposition to H2S and a-keto-butyrate. Snake venom L-amino acid oxidase. Due to the possibility that other enzymes were associated with the particulate-bound bacterial L-amino acid oxidase we compared the results with the bacterial enzyme to those obtained with pure crystalline snake v e n o m L-amino acid oxidase. The snake enzyme had a somewhat more limited specificity than the bacterial enzyme (Table IV). No appreciable activity was observed with any of the sulfoxides, nor was there any activity with S-adenosyl-L-methionine, homocysteie acid, cysteie acid, lanthionine, or eystathionine. As with the bacterial enzyme the stoichiometrie relation of 1:2:2 for oxygen consumption to ammonia and keto acids formation held for all substrates except homocysteine and cysteine. Here again oxygen consumption was disproportionately high. Results from chromatography of both

the resultant free keto acids and their dinitrophenylhydrazones were similar to those obtained with the bacterial enzyme. Optimal pH for enzymatic reactions. The effect of p H on the rate of oxidation of sulfur amino acids b y the bacterial enzyme is shown in Fig. 1. The p H o p t i m u m with Sribosylm-homocysteine, S-adenosyl-L-homocysteine, S-adenosyl-L-methionine, and Lmethionine was between 7.4 and 7.8, whereas the p H o p t i m u m for L-homocystine and djenkolic acid was 8.5-8.8. The p H optim u m for the free sulfhydryl compounds could not be measured as a function of oxygen consumption, since autooxidation of these compounds increased markedly with increasing pH. The rate of keto acid formation from these compounds was also found to increase throughout the p H range tested. The effect of p H on the rate of oxidation of sulfur amino acids b y snake v e n o m enzyme is shown in Fig. 2. The shape of the curves and p H o p t i m u m varied with respect to the individual amino acids. With some of the compounds; i.e., L-cysteine and S-adenosyl-L-homocysteine, there appeared to be two optima, one in the lower region between

L-AMINO ACID OXIDASE

59

TABLE IV 0.I0

I:~ATES OF OXYGEN CONSUMPTION AND PRODUCT ~ORMATION FROM SULFUR AMINO ACIDS BY S N A K E VENOM L-AMINO ACID OXIDA_SE a Specific activityb Substrate

S-Adenosvl-L-homocyssteine S-Ribosyl-L-homocysteine L-Methionine L-Homocvsteine L-Homocystine L-Cysteine L-Cystine L-Djenkolic acid

"~. 0.06

~/

=o 0.04

~

0~

Keto acid pro[uced

NHs

H2S

a. 0.02 ":2_ 0

1.2

2.8

2.5

0

o

2.1

4.4

4.2

0

5.6 4.1 1.4 5.7 4.1 1.3

:1.4 11.6 0 1.4 1.4 0.2 2.5 2.1 0 1.5 1.4 0.1 6.0 3.9 Trace 2.9 3.0 0

Oxygen consumption was measured manometrically. The vessels contained 15 #moles substrate, 300 ~moles phosphate buffer, pH 7.5, 300 units catalase, 250 ~moles KC1, and 25 ~g crystalline snake venom L-amino oxidase. After 30-min incubation at 30 ~ the reaction mixtures deproteinized with TCA and H~S and keto acid measured as outlined under Methods. Ammonia was measured in a duplicate vessel by diffusion technique of Braganca et al. No endogenous activity was observed. b Activity is expressed as ~moles oxygen consumed or products produced per rain/rag protein. p H 7.2-7.5 a n d t h e o t h e r a b o v e 8.5. H o m o c y s t e i n e a n d c y s t i n e e x h i b i t e d single p H o p t i m u m a t p H 8.0 a n d 8.4, r e s p e c t i v e l y . The curves with S-ribosyl-homocysteine, d j e n k o l i c acid, a n d h o m o c y s t i n e i n c r e a s e d b e y o n d p H 8.5; however, i t was difficult to e x t e n d these curves b e y o n d this p o i n t since m o s t of t h e c o m p o u n d s are alkaline labile. Determination of Michaelis constants. T h e d o u b l e - r e c i p r o c a l p l o t using b a c t e r i a l La m i n o oxidase a n d S - a d e n o s y l - L - h o m o c y s teine as s u b s t r a t e is shown in Fig. 3. T h e K ~ v a l u e for S - a d e n o s y l h o m o c y s t e i n e was 1.4 X 10 -2 M a n d t h e V . . . . was 0.26 ~ m o l e s / min/mg protein when the initial reaction velocities were expressed as t h e r a t e of k e t o a c i d f o r m a t i o n . W h e n e x p r e s s e d as t h e r a t e of o x y g e n c o n s u m p t i o n t h e K ~ was t h e s a m e a n d t h e Vm~ was e x a c t l y one-half. T h i s rel a t i o n s h i p h e l d for b o t h e n z y m e s w i t h all

0 ~ - - 0 Methionine

0.08

~ ~O~c2_S-ribosyI-L- horn 0_ .~,,~-A~^ ~S-qdenosyI-L-homo-z~.~ _ ~ystein,

L

$-adenosyI-Lmethionine

f

0.12

Homoeysteia~

0.10 !

0.08 o

0.06

0.04

~0.02

7:0

81o

r

FINAL pH

FIG. 1. Influence of pH on the rate of oxidation of sulfur amino acids by a bacterial L-amino acid oxidase. Oxygen consumption was measured polarographically. The vessel contained 5 ~rnoles substrate, 100 ~moles phosphate buffer, and 3.0 mg enzyme protein per ml. S-Ribosyl-L-homocysteine interfered with the Clark electrode and was measured manometrically. Keto acids were measured by a discontinuous assay. Vessels containing substrate and other components as described above were incubated for 5, 10, 15, and 30 min. After deproteinization, keto acids were determined with 2,4-dinitrophenylhydrazine assay, and the rates were plotted as a function of time. s u l f u r a m i n o acids t e s t e d e x c e p t h o m o c y s t e i n e a n d cysteine. A s u m m a t i o n of t h e K ~ a n d Vm~x v a l u e s o b t a i n e d with b o t h e n z y m e s a t p H 7.5 is g i v e n in T a b l e V. T h e K,, v a l u e s for cyst e i n e a n d h o m o c y s t e i n e are n e a r l y t h e s a m e for b o t h enzymes, w h e r e a s t h e Km v a l u e s for t h e o t h e r s u b s t r a t e s t e n d to b e lower w i t h t h e s n a k e e n z y m e . I n general, t h e e n z y m e f r o m b o t h sources h a d a g r e a t e r affinity for t h e free s u l f h y d r y l s a n d thio-ethers, p a r t i c u l a r l y m e t h i o n i n e . A m a r k e d decrease in affinity was o b s e r v e d b y t h e a d d i t i o n of a side chain; i.e., ribose or adenosine w i t h a f u r t h e r decrease w h e n t h e t h i o - e t h e r w a s o x i d i z e d to t h e sulfoxide.

<60

CHEN, WALGATE, AND DUERRE

S-adenosyl-L-hor n o - ~ S_r~bosvl_L_Homo_

o;.,.,o.

I0.0

FOR BACTERIAL AND SNAKE VENOM L-AMINO ACIDS OXIDASESa

E .g "~ o

ao 6.0

"~

4.0

~

2,0

o

Bacteria

10.0

i

.

i

,

L-methionine

~

8.0 E 6.0

!~// ~

C,

4.0

--~ 2.0 0

Snake venom

Substrate

0 ~

TABLE V Km AND ]7. . . . VALUES FOR SULFUR AMINO ACIDS

~

~

L

-

.... ystein,a

h

~

~

X

L-h .... ystin,

L cystr

7.0

8.0 FINAL pll

I 9.0

FIG. 2. Influence of pH on rate of oxidation of sulfur amino acids by snake veuom L-amino acid oxidase. Oxygen consumption and keto acids were measured as outlined in Fig. 1. Vessels contained 10 ~moles substrate, 103 ~moles phosphate buffer, 80 umoles KC1,100 units beef liver catalase, and 2 t~g enzyme per nil. 40

2~I i I I fl -l.0 0 ]0 2.0 3.0 (5-ADENOSYL-L-HOMOCYSTEINE• IOZM)~ F1o. 3. Double-reciprocal plot of i n i t i a l reaction velocities against S-adenosyl-L-homocysteine

concentrations expressed as the rate of oxygen consumption (C O ) and keto acid formation ( X - - X ) . The oxygen consumption was measured at 30~ using the Gilson oxygraph. The reaction vessel contained varying concentrations of substrafe, 150 /~m01e phosphate buffer, ptt 7.5, and 0.18 rag enzyme protein in the final volume of 1.5 nil. Keto acids were measured by 2,4-dinitrophenylhydrazine assay. Duplicate sets of tube contained varying concentrations of substrate, 150 tunole phosphate buffer, pH 7.5, and 1.5 mg enzyme protein in final volume of 1.5 ml. One set of tubes was incubated at 30~ for 15 mid and the other for 30 rain. The initial reaction velocities were d e termiDed by plotting the rate of keto acid formation against time and extrapolating to zero time.

J,-Homocysteine L-Cysteine l,-Methionine L-Homocystine S-Ribosyl-L-homocyst eine S-Adenosyl-L-homocysteine S-Adenosyl-L-methio: nine 5/Iethionine sulfoxide Djenkolic acid ~-Adenosyl-L-homocysteine sulfoxide L-Cystine

Km Vma~ :raM)

Vmax

1.31 5.3 9.6 12.5 13.0

0.03 0.01 0.42 1.43 0.12

5.9 2.7 20.0 7.3 18.1

14.0 0.26

11.6

14.0 0.24 45.0 0.03 83.01 0.16 00.0 0.06

18.4

__b

31.0

The values were determined from the reciprocal plots of the initial reaction velocities (keto acid formation) against reciprocals of molar concentration of substrate. The rates of keto acid formation were determined as outlined under Figs. 1 and 2. b Activity was too low for accurate K,~ deterrainations. DISCUSSION T h e L-amino acid oxidases from b o t h snake v e n o m a n d a b a c t e r i u m have b e e n f o u n d to catalyze the oxidative d e a m i n a t i o n of several sulfur a m i n o acids. T h e degree of a c t i v i t y a n d affinity t o w a r d the different s u b s t r a t e s was f o u n d to differ b e t w e e n the two e n z y m e s as well as t h a t r e p o r t e d for the e n z y m e from o t h e r sources. T h e e n z y m e from mollusca has b e e n shown p r e v i o u s l y to oxidize cystathio n i n e (10) whereas b o t h e n z y m e s discussed here did not. T h e L-amino acid oxidase from Proteus vulgaris has b e e n shown to h a v e no m e a s u r a b l e a c t i v i t y on either cysteine or c y s t i n e (3), while the e n z y m e from Proteus rettgeri was r e l a t i v e l y active t,ow~rd b o t h these substrates. I n general, the e n z y m e from b o t h sources h a d a greater affinity for the s u l f h y d r y l a n d thio-ether compounds, particularly methionine, with a m a r k e d decrease i n affinity b y the a d d i t i o n of a side chain, i.e., ribose or

L-AMINO

ACID

adenine, with the lowest affinity and activity when the thioether was oxidized to the sulfoxide. The bacterial enzyme was quite active with the sulfonium compound, S-adenosylmethionine. The compound is relatively resistant to attack by most enzymes, including the snake venom L-amino acid oxidase from Crotalus terrificus terrificus. The possibility arose that under alkaline pH suitable for the oxidation to occur, the substrate may decompose chemically or enzymaticMly. The result from paper chromatography of the reaction mixture showed that decomposition did occur with the resultant formation of methylthioadenosine and homoserine. However, no measurable activity was found with homoserine. The mechanism for the liberation of hydrogen sulfide from the free sulfhydryl compounds cysteine and homoeysteine was

OXIDASE

61

ase, whereby the sulfur liberated is reduced to hydrogen sulfide by a mercaptan or glutathione (30). However, neither mercaptoethanol nor glutathione (5 raM) had any effect on the production of keto acids or H2S from either homoeysteine or eysteine. The most plausible explanation as illustrated below is that the mercapto-a-keto acid produced from cysteine or homocysteine by an L-amino acid oxidase undergoes spontaneous decomposition under the conditions employed. Spontaneous decomposition of ~-mercaptopyruvate has been reported previously (31). Similarly it was observed that the rate of formation of hydrogen sulfide from r was not affected by either the highly purified snake venom oxidase nor the bacterial partieulate fraction and that spontaneous decomposition could account for the amount of hydrogen sulfide produced. Such findings

L-amino acid oxidase 02 HzOz spontaneous ,

H H L- cysteine

NH 3

> HzS

r162

+

H-

-

-COOH

I

H ,8- mercaptopyruvate

studied extensively. Nonoxidative deamination of serine, threonine, cysteine and homoeysteine, and diearboxylic amino acids has been shown by a group of pyridoxal phosphate-dependent enzymes. Several pyridoxal phosphate-dependent enzymes, in eluding eysteine desulfhydrase, cystathionase, and tryptophanase, catalyze the conversion of cysteine to pyruvate, ammonia, and hydrogen sulfide (29). Except for the lower yield of H2S the resultant products are the same as those catalyzed by the bacterial and snake venom L-amino acid oxidases used in this study. It is possible that the bacterial particulate fraction contained one of these enzymes. However, no activity was observed under anaerobic conditions nor did pyridoxal phosphate stimulate the reaction; hence, the presence of one of these enzymes is unlikely. Similarly the particulate fraction employed could contain a mereapto-a-keto transfer-

H 0 H pyruvate

might make it difficult to interpret results on hydrogen sulfide production from cysteine or homocysteine by other enzymes unless they are free of detectable oxidase activity. The oxidation of the free sulfhydryl group of cysteine and homocysteine to their respective disulfides and acids in the prescenee of the bacterial or snake venom L-amino acid oxidases is not understood. The oxidation of the sulfhydryl group cannot be accounted for through spontaneous oxidation; for under the experimental conditions employed, little or no oxygen uptake was observed in the absence of added enzymes. The oxidation products formed from homocysteine and cysteine do not appear to be due primarily to the hydrogen peroxide formed in the oxidative deamination reaction. The amount of nascent catalase present in the bacterial preparation and that added to the snake enzyme was sufficient to degrade any

CHEN, WALGATE, AND DUERRE

62

peroxide formed quite rapidly. Furthermore, no significant increase in oxygen consumption was observed when homocysteine, eysteine, or ~-mercaptopyruvate was added to a reaction vessel containing glucose, glucose oxidase, and catalase. F r o m the experimental evidence available it appears t h a t the oxidation of the free sulfhydryl group m a y be accelerated when the compound is bound to the L-amino acid oxidase or t h a t the enzyme preparation employed m a y have contained an enzyme which catalyzed the oxidation of this group. The oxidative deamination of the disulfide compound, homocystine, was also investigated. Since this compound contains two asymmetric centers, selective oxidation of either of the two amino groups should not oeeur. W h e n the oxidation of homocystine was followed to completion, a stoichiometrie relation of 1 mole of oxygen per mole substrate utilized was observed. This would indicate that both amino groups were oxidized. T h a t the ultimate reaction product was a diketo derivative was confirmed b y chemical analysis. Although the m o n o - a m i n o m o n o - k e t o derivative was not isolated in quantities sufficient for complete analysis, the chemical properties of one of the compounds found early in the reaction would H H H I

!

I

H H - S- S

NH 2 H H

H

- COOH H H NH ' 2

9

L-omino or oxidase

H

H

HOOC-

H

s- s

~)

-COOH i

H H

H

I

i

H NH~)

L-omino ocid

1//~H2 02 O

H

H

H

oxidose

H

HOOC-C-C-C-S-S-C-C-C-COOH ii

O

i

I

H H

i

H

I

ii

H O

suggest the presence of such an intermediate. The following reaction sequence has been proposod for the oxidative deamination of homoeystine. We did not carry out a detailed analysis of the product or products derived from dj enkolic acid. However. as with homocystine when the reaction was followed to completion a stoiehiometric relationship of 1 mole of oxygen per mole substrate utilized was observed. This would indicate t h a t the final reaction product was 3 , 3 ' m e t h y l e n e dithiobis (2-ketopropanoic acid). REFERENCES 1. BUI~TON,K., Biochem. J. 50,258 (1952). 2. KNIGHT,S. G., J. Bacteriol. 55,401 (1948). 3. STUraPF, P. K., AND GREEN, D. E., J. Biol. Chem. 153,387 (1944). 4. ZELLER, E. A., AND MARITZ, A., Helm Chim. Acta 27, 1888 (1944). 5. ZELm.'R,E. A., Advan. Enzymol. 8, 459 (1948). 6. BLANCHARD,M., GREEN, D. E., •OCITO, V., AND RATNER, S., J. Biol. Chem. 155, 421

(1944). 7. NAKANO,M., AND DANOWSKI, T. S., J. Biol. Chem. 241, 2075 (1966). 8. BOULANGER, P., AND OSTEUX, I~., Biochem. Biophys. Acta 21, 552 (1956). 9. STRUCK,J., AND SIZER, I. W., Arch. Bioehem. Biophys. 90, 22 (1960). 10. BLASCHKO,H., AND HOPE, D. B., Biochem. J.

62,335 (1956). 11. RoeaE, J., GLAHN, P., MANCItON, P., AND THOAI, N., Biochem. Biophys. Acta 35, 111 (1959). 12. THAYER, P. S., AND HOROWITZ, N. H., J. Biol. Chem. 192,755 (1951). 13. GREENS'rEIN, J. P., BIRNBAUM, S. M., AND 0TEY, M. C., J. Biol. Chem. 204,307 (1953). 14. MILLER, C. H., AND DUERRE, J. A., J. Biol. Chem. 244, 4273 (1969). 15. DUERRE, J. A., AND MILLER, C. H., Anal. Biochem. 17,310 (1966). 16. DUERRE, J. A., SALISBURY, L., AND MILLER, C. It., Anal. Biochem. 35,505 (1970). 17. SCItLENK, F., ])AINKO, J. L., AND DEPALMA, R. E., Arch. Bioehem. Biophys. 83, 28 (1959). 18. KUN, E., Biochem. Biophys. Acta 25,137 (1957). 19. ])UERRE, J. A., AND BUCKLEY, P. J., J. Bacteriol. 90, 1686 (1965). 20. DOWELL, V. K., JR., AND HAWKINS, T. M., "Laboratory Methods in Anaerobic Bacteriology," U.S. Government Printing Office, Washington, D.C., 1968. 21. KRISHNAMURTHI,V. S., BUCKLEY, P. J., AND

L-AMINO

22. 23. 24. 25.

ACID

DUERRE, J. A., Arch. Biochem. Biophys. 130, 636 (1969). Low~Y, O. H., ROSEBROVG~, N. J., FARR, A. L., AND I~A_NDALL,R. J., J. Biol. Chem. 193, 265 (1951). FRIEDEMANN, T. E., AND HAUGEN, G. E., J. Biol. Chem. 147,415 (1943). BRAGANCA, B. M., QVASTEL, U. H., AND SCHUCHER, 1~., Arch. Biochem. Biophys. 52, 18 (1954). BADINGS, H. T., AND VAN DEn POL, J. J. G., Neth. Milk Dairy J. 19,283 (1965).

26. UMBREIT, W. W., BURRIS, R. H., AND STAIJF-

0XIDASE

27. 28. 29. 30. 31.

63

FER, J. F., "Manometric Techniques," Burgess, Minneapolis, 1964. DANClS, J., ttUTZLER, J., AND LEVITZ, M., Biochem. Biophys. Acta 78, 85 (1963). DUERRE, J. A., MILLER, C. H., AND REAMS, G. G., J. Biol. Chem. 244,107 (1969). MEISTER, A., "Biochemistry of Amino Acids," Vol. 2, pp. 793, 879. Academic Press, New York, 1965. MEISTER, A., FRASER, P. E., AND TICE, S. V., J. Biol. Chem. 206,561 (1954). KONDO, Y., KA~EVAMA, T., AND TAMIYA,N., J. Biochem. Tokyo 43,749 (1956).