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[ O/ O] P-NMR Enzymes
Studies of Phosphoryl Transfer
J. J. VILLAFRANCA, F. M. RAUSHEL, C. DEBROSSE, and T. D. MEEK
R.
P.
PILLAI,
M. S.
BALAKRISHNAN,
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802
In 1978 Cohn and Hu (1) demonstrated the isotopic effect by 0 on the P-nmrspectrum of inorganic phosphate. For each 0 that replaces an 0, an upfield shift of 0.021 ppm results. Thus the chemical shift forHP 0 =is 0.084 ppm downfield from HP180=. Based on this observation it is obvious that any chemical event that potentially involves substitution or exchange of an 0 for 0 in phosphorus containing compounds can be followed by monitoring the P-nmrspectrum throughout the course of the reaction. This article describes experiments from my laboratory on two enzymes that catalyze phosphoryl transfer reactions and our use of the [ 0/ ] p-nmr methodology to detect intermediates in the enzymic reactions. Glutamine synthetase catalyzes the formation of glutamine from ATP, gluatmate, and ammonia. The other products are ADP and The reaction mechanism is though to proceed through a γ-glutamyl phosphate intermediate. We have shown that incubation of ADP, glutamine, and [ 0] i with glutamine synthetase resulted in the loss of 0 from the i (2). Analysis of the data showed that only one 0 was lost per encounter and that the rate constant for exchange was 5-7 times faster than net turnover of products. This was also demonstrated by Stokes and Boyer (3) using mass spectrometry. 18
31
18
16
16
4
4
18
16
31
18
160
31
18
18
p
P
18
CL #-l Η· — Ρ - · " I ·"
+
t
I R
I R
NH,
{
(IÎ
II I R
+ ADP
I R
+
II ADP-O-P-t" I t"
(I)
0097-6156/81/0171-0131$05.00/0 © 1981 American Chemical Society
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
132
PHOSPHORUS CHEMISTRY
T h i s exchange r e a c t i o n i s explained by assuming that the above r e a c t i o n s are o c c u r r i n g on the enzyme s u r f a c e and that the γ-carboxyl of glutamate ( I I ) i s f r e e t o r o t a t e , thus a l l o w i n g , upon r e v e r s a l of the r e a c t i o n , the formation of γ-glutamyl phosphate ( I ) with an ^ 0 ± the bridge p o s i t i o n . Reaction of I with NH3 would then produce V± with only 3 atoms of ^ 0 i n s t e a d of the o r i g i n a l 4. Since a random d i s t r i b u t i o n of ^ 0 ± maintained a t a l l times, only one ^ 0 i s l o s t per encounter with the enzyme and thus must be d i s s o c i a t i n g from the enzyme f a s t e r than glutamine. Recently M i d e l f o r t and Rose (4_) introduced a p o s i t i o n a l i s o t o p e exchange technique that i s a l s o s u i t a b l e f o r study by the •^0 chemical s h i f t technique. B r i e f l y , t h i s technique f o l l o w s the exchange of l a b e l from one part of a s u b s t r a t e to another due t o r o t a t i o n a l equivalence of some i n t e r m e d i a t e . The method was f i r s t a p p l i e d t o glutamine synthetase i n the r e a c t i o n : n
Q
0 0 0 11 11 11 Ado-0-P-0-P-#-P-0~ 1 I I
o~
cr
or
0 0 11 % » + Glu = = è A d o - 0 - P - 0 > P - t + χ - G l u t o m y l - Ρ I I %
ο" o"
"rotation"
0 · II II A d o - O - P - O - P - 0 " + y-Glutomyl-P 7=^ I I 0" 0" v
0 · 0 II II II A d o - 0 - P - 0 - P - 0 - P - 0 ~ + Glu I I I 0" 0" 0"
ATP i s synthesized with 0 i n the 3 - γ bridge p o s i t i o n . In phosphate-containing species such as ATP where the phosphorus ( a , 3 , and γ) and oxygen (bridge and nonbridge) atoms are i n d i f f e r e n t environments the e f f e c t of ^ 0 s u b s t i t u t i o n on the 31p chemical s h i f t i s dependent on the oxygen environment. The s h i f t i s l a r g e s t f o r those oxygens with most double bond c h a r a c t e r . For the 3-P, the s h i f t per 0 atom i s 0.016 ppm f o r the 3 - γ bridge p o s i t i o n and 0.028 ppm f o r the 3 nonbridge p o s i t i o n . Presuming glutamine synthetase c a t a l y z e d the formation of γ-glutamyl-P from ATP and glutamate i n the absence of N H 3 , then ADP would be formed; because of r o t a t i o n a l equivalence i t would allow, upon the r e v e r s a l of the above r e a c t i o n s , the formation of ATP with 0 i n the nonbridge p o s i t i o n of the 3~Ρ· M i d e l f o r t and Rose found t h i s t o be the case f o r glutamine synthetase by mass s p e c t r a l a n a l y s i s of the 18O/16Q exchange and we have followed t h i s r e a c t i o n by nmr. We detected the l o s s of 0 from the γ-Ρ s i g n a l (the s t a r t i n g m a t e r i a l had 0 i n a l l four oxygens of the γ-Ρ) and the s h i f t of s i g n a l from the 3~Ύ bridge to 3 nonbridge p o s i t i o n as p r e d i c t e d above. Thus a l l l i n e s of evidence p o i n t t o formation of γ-glutamyl-P as a k i n e t i c a l l y competent intermediate i n c a t a l y s i s . 1 8
1 8
1 8
1 8
1 8
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
25.
VILLAFRANCA ET AL.
Phosphoryl
Transfer Enzymes
133
Carbamyl phosphate synthetase c a t a l y z e s the synthesis of carbamyl-P from HCO3"» glutamine, and 2 moles of ATP. The enzyme a l s o c a t a l y z e s the HC03""-dependent h y d r o l y s i s of ATP. Raushel and V i l l a f r a n c a (5) followed the exchange of 1^0 from the bridge to the nonbridge p o s i t i o n of [γ-18ο]ΑΤΡ a f t e r i n c u b a t i o n with enzyme and bicarbonate. The exchange r a t e was 0.4 times the r a t e of ADP formation. These r e s u l t s support the formation of carboxy phosphate as the f i r s t intermediate i n the c a t a l y t i c sequence. The r a t e of formation of intermediates i n the r e a c t i o n was a l s o s t u d i e d using r a p i d r e a c t i o n techniques by Raushel and V i l l a f r a n c a (6) and the data agree with the ^Ip-nmr s t u d i e s presented above. The p o s i t i o n a l isotope exchange has a l s o been measured with 31p-nmr i n the reverse r e a c t i o n of carbamyl phosphate synthetase : Carbamyl-P + ADP
+ ATP + NH3
+ HCO3".
Carbamyl phosphate was synthesized with 1^0 i n a l l oxygens except the carbonyl oxygen of carbon. The exchange of the bridge oxygen i n t o the carbonyl oxygen was followed by nmr and was evidence f o r the f o l l o w i n g s e r i e s of r e a c t i o n s
Ν Η
2
0 · II II - 0 - · - Ρ - · ~
Ο II ADP
4-
ADP
•·
II II N H - C - 0 - P - # ~ I 2
r
+
ATP
NH -C-CT
+
ATP
NH 4-
9
2
t"
These data support the formation of a second intermediate i n the r e a c t i o n pathway, v i z , carbamate (NH2CO2"") and i t s formation i s ~4 times f a s t e r than ATP formation f o r the reverse r e a c t i o n o u t l i n e d above. In c o n c l u s i o n the [18rj/16o] 3 1 p method i s a powerful technique f o r the study of r e a c t i o n intermediates i n phosphoryl t r a n s f e r r e a c t i o n s . Both the nature of the r e a c t i v e species as w e l l as the r a t e of formation and breakdown of the r e a c t i v e species on the enzyme can be e s t a b l i s h e d . Work supported by NIH grants AM-21785, AM-05996, GM-23529 and NSF Grant PCM-7807845. n m r
Literature Cited
1.
Cohn, M.; Hu, A. Proc. Natl. Acad. Sci. USA 1978, 200-3.
75,
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
134
PHOSPHORUS CHEMISTRY
2. Balakrishnan, M. S.; Sharp, T. R.; Villafranca, J. J. Biochem. Biophys. Res. Commun. 1978, 85, 991-8. 3. Stokes, B. O.; Boyer, P. D. J. Biol. Chem. 1976, 251, 5558-64. 4. Midelfort, C. F.; Rose, I. A. J. Biol. Chem. 1976, 251, 5881-7. 5. Raushel, F. M.; Villafranca, J. J. Biochemistry 1980, 19, 3170-4. 6. Raushel, F. M.; Villafranca, J. J. Biochemistry 1979, 18, 3424-9. R E C E I V E D June 30, 1981.
Quin and Verkade; Phosphorus Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
