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The product inhibition of sulphatase B
365
SHORT COMMUNICATIONS
BBA 63467
The product inhibition of sulphatase B I t was reported some years ago z that sulphatase B (aryl-sulphate sulphohydrolase, EC 3.1.6.1) from ox liver was noncompetitively inhibited b y sulphate, one of the reaction products. Other workers z suggested that the inhibition was competitive, although they could not confirm this b y kinetic measurements. Because of the importance of studies of product inhibition in elucidating the mechanism of enzyme reactions, this problem has been reinvestigated using a purified specimen of sulphatase B. The enzyme was a mixture of the isoenzymes Ba and Bfl which do not differ in their kinetic properties 3. Sulphatase B (specific activity 7 ° units/rag) was prepared and assayed spectrophotometrically as previously described a. Kinetic constants were calculated by the method of WILKINSON 4.
16
10. -2.0
12
8
4
, J 0
0.5
1.0
1.5
, 5
o
2D
Sulphate
Y[S] (mM-')
concn.
, 1o
(raM)
Fig. i, Reciprocal plots s h o w i n g t h e effect of s u l p h a t e on t h e a c t i v i t y of s u l p h a t a s e B. S u b s t r a t e , n i t r o c a t e c h o l s u l p h a t e ; p H 5-4 in 0. 5 M s o d i u m a c e t a t e - a c e t i c acid buffer; inhibitor, K , S O , at t h e c o n c e n t r a t i o n s s h o w n on t h e figure; i n c u b a t i o n , 5 m i n a t 37 °. Fig. 2. R e p l o t s of t h e slopes (O) a n d i n t e r c e p t s on t h e o r d i n a t e ( I ) concentration.
of Fig. i a g a i n s t s u l p h a t e
The raw data are shown in Fig. I which clearly demonstrates the noncompetitive nature of the inhibition. The intersection of the lines above the abscissa shows that K, calculated from the slopes of the lines must be less than that calculated from the intercepts on the ordinate 5. Fig. 2 shows replots of the slopes and intercepts from Fig. I and gives values of 1.2 mM and 4.6 mM SO42-, respectively, for K, slope and K, intereept. The plots in Fig. 2 also show that the inhibition is linear noncompetitive 5. The simplest reasonable mechanism for a hydrolytic enzyme is that of an ordered uni-bi reaction : E 4- S .~ E S ~- E P z P 2 ~ E P a +
P1 ~
E + P
The inhibition of such a reaction by P1 is linear noncompetitive with K, slove and K / i n t e r e e p t being, in general, different, whereas the inhibition b y P , is linear competitive. Assuming the reaction catalysed by sulphatase B to be of the above form, the present findings show that sulphate cannot be the last-released product although B i o c h i m . B i o p h y s . A c t a , 198 (t97 c) 365-366
366
BIOCHIMICA ET BIOPHYSICA ACTA
it could be the first-released: this means that E P 2 must be a sulphatase B-phenol c o m p l e x . T h e i n h i b i t i o n o f s u l p h a t a s e A b y s u l p h a t e is, o n t h e o t h e r h a n d , l i n e a r c o m p e t i t i v e (ref. 6 a n d p e r s o n a l c o m m u n i c a t i o n f r o m D r . R. G. NICI~OLLS) a n d t h e r e f o r e c o n s i s t e n t w i t h t h e l a s t - r e l e a s e d p r o d u c t b e i n g s u l p h a t e a n d EP2 b e i n g a s u l p h o s u l p h a t a s e A. C o n f i r m a t i o n o f t h e s e p o s t u l a t e d m e c h a n i s m s c o u l d b e o b t a i n e d by determining the type of inhibition caused by the other product of the arylsulphatase reactions, a phenol (4-nitrocatechol in the present instance). Unfortunately this f u r t h e r i n f o r m a t i o n is u n a v a i l a b l e b e c a u s e n e i t h e r s u l p h a t a s e A n o r B is i n h i b i t e d b y 4 - n i t r o c a t e c h o l a t t h e h i g h e s t c o n c e n t r a t i o n , o.6 m M , p r a c t i c a b l e t o u s e in a s p e c t r o photometric assay. T h e s e r e s u l t s n e v e r t h e l e s s s h o w t h a t t h e r e is a n i m p o r t a n t d i f f e r e n c e b e t w e e n t h e t y p e s o f r e a c t i o n c a t a l y s e d b y s u l p h a t a s e s A a n d B, a n d it s e e m s t h a t t h e difference between these enzymes may be more fundamental than has been suggested in t h e p a s t a,7.
Department of Physical Biochemistry, John Curtis School of Medical Research, Australian National University, Canberra (Australia) i 2 3 4 5 6 7
A. B. R o Y
A. B. RoY, Biochem. J., 57 (1954) 465 • E. C. WEBB AND P. F. W. MOnROW, Biochem. J., 73 (1959) 7. E. ALLEN AND A. B. ROY, Biochim. Biophys. Acta, 168 (1968) 243. G. N. WILKINSON, Biochem. J., 80 (1961) 324 . W. W. CLELAND, Biochim. Biophys. Acta, 67 (1963) lO4, 173. A. B. RoY, Biochem. J., 55 (1953) 653. H. BAUM AND K. S. DODGSON, Biochem. J., 69 (1958) 573.
R e c e i v e d M a r c h 2 4 t h , 1970
Biochim. Biophys. Acta, 212 (197 o) 365 366
TITLES
OF RELATED
PAPERS
IN OTHER
SECTIONS
The following papers t h a t have recently appeared in other sections of BIOCHIMICA ET BIOPHYSICA ACTA may be of interest to the readers of this specialized section : B B A - G E N E R A L SUBJECTS Enzymatic synthesis of glycerol i-phosphate. Elevation in diabetic and fasted animals, compared with glucose-6-phosphatase and related enzyme activities (BBA 26349) by M. R. STI~TTEN (New Brunswick, N.J.) . . . . . . . . . . . . . . 208 (I97 o) Glycogen-bound phosphorylase in Cryptococcus laurentii (BBA 26363) by J. C. SCHOLTZ AND H. ANKEL (Milwaukee, Wisc.) . . . . . . . . . 215 (197 o) The in vivo effect of vitamin E in experimental porphyria (BBA 26389 ) by P. P. NAIR, H. S. MURTY AND N. R. GROSSMAN (Baltimore, Md.) . . 215 (197 o) The distribution of asparaginase activity in legumes (BBA 26351) by E. M. LEES AND A. B. BLAKENEY (Sydney) . . . . . . . . . . . . 215 (197 o) Anlino-acid oxidase action on D- and L-a,y-diaminobutyric acid (BBA 23597) by C. H. CHEX AND R. E. KOEPPE (Stillwater, Okla.) . . . . . . . . . 215 (197 o) Lack of hydroxylation-induced migration with 4-iodophenylalanine (BBA 23598 ) by R. E. COONSELL, P. S. CHAN AND P. A. WEINHOLD (Ann Arbor, Mich.) 215 (197 o) Enzymatic ethanolysis of uridine 5t-monophosphate (BBA 23590) by J. BucHOWlCZ AND A. LE~NIEWSKA (Warsaw) . . . . . . . . . . . 215 (197 o)
394 39 112 145 184 187 198
SHORT COMMUNICATIONS
BBA 63467
The product inhibition of sulphatase B I t was reported some years ago z that sulphatase B (aryl-sulphate sulphohydrolase, EC 3.1.6.1) from ox liver was noncompetitively inhibited b y sulphate, one of the reaction products. Other workers z suggested that the inhibition was competitive, although they could not confirm this b y kinetic measurements. Because of the importance of studies of product inhibition in elucidating the mechanism of enzyme reactions, this problem has been reinvestigated using a purified specimen of sulphatase B. The enzyme was a mixture of the isoenzymes Ba and Bfl which do not differ in their kinetic properties 3. Sulphatase B (specific activity 7 ° units/rag) was prepared and assayed spectrophotometrically as previously described a. Kinetic constants were calculated by the method of WILKINSON 4.
16
10. -2.0
12
8
4
, J 0
0.5
1.0
1.5
, 5
o
2D
Sulphate
Y[S] (mM-')
concn.
, 1o
(raM)
Fig. i, Reciprocal plots s h o w i n g t h e effect of s u l p h a t e on t h e a c t i v i t y of s u l p h a t a s e B. S u b s t r a t e , n i t r o c a t e c h o l s u l p h a t e ; p H 5-4 in 0. 5 M s o d i u m a c e t a t e - a c e t i c acid buffer; inhibitor, K , S O , at t h e c o n c e n t r a t i o n s s h o w n on t h e figure; i n c u b a t i o n , 5 m i n a t 37 °. Fig. 2. R e p l o t s of t h e slopes (O) a n d i n t e r c e p t s on t h e o r d i n a t e ( I ) concentration.
of Fig. i a g a i n s t s u l p h a t e
The raw data are shown in Fig. I which clearly demonstrates the noncompetitive nature of the inhibition. The intersection of the lines above the abscissa shows that K, calculated from the slopes of the lines must be less than that calculated from the intercepts on the ordinate 5. Fig. 2 shows replots of the slopes and intercepts from Fig. I and gives values of 1.2 mM and 4.6 mM SO42-, respectively, for K, slope and K, intereept. The plots in Fig. 2 also show that the inhibition is linear noncompetitive 5. The simplest reasonable mechanism for a hydrolytic enzyme is that of an ordered uni-bi reaction : E 4- S .~ E S ~- E P z P 2 ~ E P a +
P1 ~
E + P
The inhibition of such a reaction by P1 is linear noncompetitive with K, slove and K / i n t e r e e p t being, in general, different, whereas the inhibition b y P , is linear competitive. Assuming the reaction catalysed by sulphatase B to be of the above form, the present findings show that sulphate cannot be the last-released product although B i o c h i m . B i o p h y s . A c t a , 198 (t97 c) 365-366
366
BIOCHIMICA ET BIOPHYSICA ACTA
it could be the first-released: this means that E P 2 must be a sulphatase B-phenol c o m p l e x . T h e i n h i b i t i o n o f s u l p h a t a s e A b y s u l p h a t e is, o n t h e o t h e r h a n d , l i n e a r c o m p e t i t i v e (ref. 6 a n d p e r s o n a l c o m m u n i c a t i o n f r o m D r . R. G. NICI~OLLS) a n d t h e r e f o r e c o n s i s t e n t w i t h t h e l a s t - r e l e a s e d p r o d u c t b e i n g s u l p h a t e a n d EP2 b e i n g a s u l p h o s u l p h a t a s e A. C o n f i r m a t i o n o f t h e s e p o s t u l a t e d m e c h a n i s m s c o u l d b e o b t a i n e d by determining the type of inhibition caused by the other product of the arylsulphatase reactions, a phenol (4-nitrocatechol in the present instance). Unfortunately this f u r t h e r i n f o r m a t i o n is u n a v a i l a b l e b e c a u s e n e i t h e r s u l p h a t a s e A n o r B is i n h i b i t e d b y 4 - n i t r o c a t e c h o l a t t h e h i g h e s t c o n c e n t r a t i o n , o.6 m M , p r a c t i c a b l e t o u s e in a s p e c t r o photometric assay. T h e s e r e s u l t s n e v e r t h e l e s s s h o w t h a t t h e r e is a n i m p o r t a n t d i f f e r e n c e b e t w e e n t h e t y p e s o f r e a c t i o n c a t a l y s e d b y s u l p h a t a s e s A a n d B, a n d it s e e m s t h a t t h e difference between these enzymes may be more fundamental than has been suggested in t h e p a s t a,7.
Department of Physical Biochemistry, John Curtis School of Medical Research, Australian National University, Canberra (Australia) i 2 3 4 5 6 7
A. B. R o Y
A. B. RoY, Biochem. J., 57 (1954) 465 • E. C. WEBB AND P. F. W. MOnROW, Biochem. J., 73 (1959) 7. E. ALLEN AND A. B. ROY, Biochim. Biophys. Acta, 168 (1968) 243. G. N. WILKINSON, Biochem. J., 80 (1961) 324 . W. W. CLELAND, Biochim. Biophys. Acta, 67 (1963) lO4, 173. A. B. RoY, Biochem. J., 55 (1953) 653. H. BAUM AND K. S. DODGSON, Biochem. J., 69 (1958) 573.
R e c e i v e d M a r c h 2 4 t h , 1970
Biochim. Biophys. Acta, 212 (197 o) 365 366
TITLES
OF RELATED
PAPERS
IN OTHER
SECTIONS
The following papers t h a t have recently appeared in other sections of BIOCHIMICA ET BIOPHYSICA ACTA may be of interest to the readers of this specialized section : B B A - G E N E R A L SUBJECTS Enzymatic synthesis of glycerol i-phosphate. Elevation in diabetic and fasted animals, compared with glucose-6-phosphatase and related enzyme activities (BBA 26349) by M. R. STI~TTEN (New Brunswick, N.J.) . . . . . . . . . . . . . . 208 (I97 o) Glycogen-bound phosphorylase in Cryptococcus laurentii (BBA 26363) by J. C. SCHOLTZ AND H. ANKEL (Milwaukee, Wisc.) . . . . . . . . . 215 (197 o) The in vivo effect of vitamin E in experimental porphyria (BBA 26389 ) by P. P. NAIR, H. S. MURTY AND N. R. GROSSMAN (Baltimore, Md.) . . 215 (197 o) The distribution of asparaginase activity in legumes (BBA 26351) by E. M. LEES AND A. B. BLAKENEY (Sydney) . . . . . . . . . . . . 215 (197 o) Anlino-acid oxidase action on D- and L-a,y-diaminobutyric acid (BBA 23597) by C. H. CHEX AND R. E. KOEPPE (Stillwater, Okla.) . . . . . . . . . 215 (197 o) Lack of hydroxylation-induced migration with 4-iodophenylalanine (BBA 23598 ) by R. E. COONSELL, P. S. CHAN AND P. A. WEINHOLD (Ann Arbor, Mich.) 215 (197 o) Enzymatic ethanolysis of uridine 5t-monophosphate (BBA 23590) by J. BucHOWlCZ AND A. LE~NIEWSKA (Warsaw) . . . . . . . . . . . 215 (197 o)
394 39 112 145 184 187 198