Carbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24933/1/0000360.pd

A. Shiels

Allen

Armstrong

Carter

Cohen

D. Hewett-Emmett

Dodgson

Elliott

Halmann

Hansen

Hewett-Emmett

Holmes

Jones

Kelley

Kennedy

King

Koester

Lindskog

Magid

Maren

N.D. Carter

Osborne

Pausch

Pocker

R.E. Tashian

Register

Riordan

S. Jeffery

Sanyal

Tashian

Tatibana

Vincent

W.R. Chegwidden

Wanders

Wistrand

Crossref

FEBS Letters

Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate

Carter, Nicholas D.

Chegwidden, W. Richard

Hewett-Emmett, David

Jeffery, S.

Shiels, A.

Tashian, Richard E.

Deep Blue Documents at the University of Michigan

AbstractCarbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle

Carter, N.D.

Chegwidden, W.R.

Hewett-Emmett, D.

Tashian, R.E.

Elsevier - Publisher Connector 

Volume 165, number 2 FEBS 1092 January 1984 
Novel inhibition of carbonic anhydrase isozymes I, II and III by 
carbamoyl phosphate 
N.D. Carter, W.R. Chegwidden*+, D. Hewett-Emmett+, S. Jeffery, A. Shiels 
and R.E. Tashian’ 
Department of Child Health, St. George’s Hospital Medical School, London SW17 ORE, *Department of 
Siological Sciences, Sheffield City Polytechnic, Sheffield SI I WB, England and +Department of Human Genetics, 
University of Michigan Medical School, Ann Arbor, MI 48109, USA 
Received 12 September 1983 
Carbamoyl phosphate has been shown to inhibit carbonic anhydrase (CA) isozymes CA I, CA II and CA 
III. This physiologically important molecule is the most potent, naturally occurring inhibitor of carbonic 
anhydrase yet found. It is also unique, among carbonic anhydrase inhibitors discovered hitherto, in that 
it inhibits the 3 isozymes with equal effect, despite their strikingly different properties. The results imply 
the participation of carbonic anhydrase in the regulation of substrate availability for the urea cycle. 
Carbonic anhydrase isozyme Carbamoyl pho~phote Carbamoyi phosphate synthetase 
Enzyme inhibition 
1. INTRODUCTION 
Carbonic anhydrase (EC 4.2.1.1) is a 
monomeric, zinc met~loenz~e which physio- 
logically catalyzes the reversible hydration of CO2. 
In addition it acts as a general acid-base catalyst 
towards a number of ‘synthetic’ substrates uch as 
p-nitrophenyl acetate [l-3]. It is abundantly pre- 
sent in a wide variety of organisms and is con- 
sidered to be of fundamental importance in several 
different processes including calcification, respira- 
tion, and transport and accumulation of H+ and 
HCO3- [4,5]. 
In mammals, birds and reptiles, 3 different 
isozyme forms, designated CA I, CA II and CA 
III, have been identified [6-81. These exhibit strik- 
ingly different properties in terms of structure, 
particularly at the active site, catalytic activity and 
inhibition parameters [6-l l]. The CA II isozymes 
have substantially the highest levels of CO2 
hydratase and esterase @-nitrophenyl acetate) ac- 
tivities. In contrast, the CA III isozymes, abundant 
in red skeletal muscle and also present in liver of 
* To whom reprint requests should be addressed 
Urea cycie 
some mammals [6,7,9,12], possess notably low ac- 
tivities, whilst additionally possessing a novel low 
acid phosphatase activity [13], which tempts 
speculation that they fulfill an as yet unknown 
physiologic~ role other than the catalysis of 
CO2-hydration. 
Consideration of the effects of the various car- 
bonic anhydrase inhibitors hitherto examined 
reveals a similarly varied picture when the 
isozymes are compared 19,101. CA II, the major 
respiratory and secretory enzyme form in animals, 
is the isozyme most sensitive to inhibition by 
heterocyclic or aromatic sulphonamides, whilst 
CA III has a relatively low affinity for such in- 
hibitors. On the other hand, inhibition by halides 
follows a different pattern, CA II being relatively 
insensitive, whilst CA I and CA III are more readi- 
ly inhibited. Exceptionally, the cyanate anion in- 
hibits CA III the most strongly of all 3 isozymes 
f9m. 
We describe here the effects of carbamoyl 
phosphate on the 3 isozymes of carbonic 
anhydrase. Carbamoyl phosphate is the first 
naturally occurring, physiologically important 
molecule found to inhibit carbonic anhydrase 
activity. 
Published by Elsevier Science Publishers 3. V. 
~1457593/84/$3.~ 0 1984 Federation of European Bi~hemical Societies 197 
Volume 165, number 2 FEBS LETTERS January 1984 
2. MATERIALS AND METHODS 
2.1. Reagents 
Carbamoyl phosphate and p-nitrophenyl acetate 
were obtained from Sigma, MO. All other reagents 
were analytical grade. 
2.2. Enzyme purification 
Human CA I and CA II were purified from red 
cell haemolysates by affinity chromatography as in 
[14], Human CA III was prepared from post- 
mortem specimens of psoas major muscle by af- 
finity chromatography followed by gel filtration as 
in fl5]. IOOr 
Bovine and chicken CA III from skeletal muscle 
were first separated from CA I and CA II isozymes 
by affinity chromatography [14] followed by 
DEAE-cellulose chromatography and gel permea- 
tion using Sephadex G-75. 
Rat liver CA III was purified as in [12]. 
2.3. Enzyme activity measurements 
I i I I J 
1.0 2.0 3.0 
Carbamyl Phosphate (mt4 1 
CO2 hydratase activity was measured using a 
pH-stat assay system comprising a PHM 64 pH 
meter, a TTT 60 titrator and a 2.5-ml ABU 80 
burette, all from Radiometer, Copenhagen. The 
assays were performed in a total volume of 10.0 
ml, thermostatted at 2°C. The reaction was 
measured in the direction of bicarbonate dehydra- 
tion, essentially as in [l&17], at pH 7.1, using 1 .O 
M sulphuric acid as titrant. The reaction mixture 
comprised 6.7 mM Na2HP04, 6.7 mM NaH2P04, 
30 mM NaHCO3 and 0.5 mM EDTA. The product 
of the reaction (CO2) was removed by continuously 
bubbling nitrogen uniformly through the sintered 
glass floor of the reaction vessel. 
Fig. 1. Effect of carbamoyl phosphate on the inhibition 
of carbonic anhydrase isozymes CA I and CA II from 
human red cell and CA III from rat liver. Enzyme activi- 
ty was measured as dehydration of bicarbonate at pH 
7.1, using a pH-stat assay system as described in the text. 
Enzyme concentrations in each set of assays were: CA I, 
90gg/ml; CA II, 0.94~g/ml; CA III, 0.38 mgiml. 
human, bovine, sheep and chicken skeletal muscle 
and rat liver. 
Esterase activity toward ~-nitrophenyl acetate 
was measured spectrophotometrically at 348 nm as 
in [IS]. The reaction mixture comprised 30 mM 
sodium diethylmalonate buffer (pH 7.2) and 1 mM 
p-nitrophenyl acetate in a total volume of 1.0 ml. 
Fig. 1 shows the effect of carbamoyl phosphate 
concentration on the inhibition of the bicarbonate 
dehydration activity of human red cell CA I and 
CA II and rat liver CA III. Clearly, the 150 of = 
0.2 mM is the same for each isozyme. 
3. RESULTS 
The .effects of carbamoyl phosphate concentra- 
tion on the esterase activity toward p-nitrophenyl 
acetate of human and sheep CA III were also ex- 
amined and similar results and ISO values were 
obtained. 
Carbamoyl phosphate inhibits both the bicar- These results show that carbamoyl phosphate is 
bonate dehydration and the esterase activities of the most potent naturally occurring inhibitor of 
the 3 isozymes of carbonic anhydrase. A concen- carbonic anhydrase yet discovered. It is con- 
tration of 25 mM achieved total inhibition of siderably more inhibitory than Cl- [19,20] and its 
human red cell CA I and CA II, CA activity in a potency of CA III inhibition is comparable to that 
total human red cell lysate, and CA III from of acetazolamide [9,21]. 
198 
Volume 165. number 2 FEBS LETTERS January 1984 
4. DISCUSSION 
The inhibition of carbonic anhydrase by car- 
bamoyl phosphate is of significant interest both to 
the molecular enzymologist and in the broader 
metabolic arena. The most striking feature, in en- 
zymological terms, is the uniform degree of inhibi- 
tion among the different isozyme forms. All 
previously published work on carbonic anhydrase 
inhibitors shows a considerable variation in degree 
of inhibition from one isozyme to another [4,10]. 
In the case of the sulphonamides, the most power- 
ful known inhibitors of carbonic anhydrase, the Ki 
values for the different isozymes range by a factor 
approaching 104, with CA III showing a 
remarkable resistance to sulphonamide inhibition 
in comparison with the other two isozymes 
[4,9,10,21]. CA II is about lo-times more sensitive 
than CA I to acetazolamide and sulphanilamide, 
but shows less difference in response to other 
sulphonamides [4,10]. Conversely, when we con- 
sider the effect of halide inhibitors, we find that 
CA is is about IOO-times more sensitive to inhibi- 
tion than CA II, and is similar to CA III in this 
respect [9, lo]. 
Considered against this background, the iden- 
tical inhibition of all 3 isozymes by carbamoyl 
phosphate is remarkable. This is especially so with 
regard to CA III, which shows fundamental dif- 
ferences in active site composition by comparison 
with the other two isozymes [ll]. Of the 5 putative 
active site residues unique to CA III, two (posi- 
tions 67, 91) are arginines, which might well be ex- 
pected to interact with phosphorylated modulators 
[22]. Thus, the equivalent inhibition of the 3 
isozymes suggests that carbamoyl phosphate may 
be binding elsewhere than at this isozyme-specific 
region of the active site. 
The uniform nature of inhibition also provides 
confirmatory evidence that carbamoyl phosphate 
is a genuine inhibitor. At alkaline pH, carbamoyl 
phosphate breaks down to give cyanate and 
phosphate [23-251. The possibility of the inhibi- 
tion observed here being due to cyanate con- 
tamination is however excluded, since this anion 
shows different degrees of inhibition with each 
isozyme [10,19]. 
The metabolic implications of carbamoyl 
phosphate inhibition are intriguing. Despite the 
difficulties of measuring tissue levels of carbamoyl 
phosphate, due to the instability of the molecule 
during extraction [26,27], intramitochondrial 
levels as high as 22 nmol - mg-r mitochondrial pro- 
tein, a level clearly inhibitory to carbonic 
anhydrase, have been recorded using a rapid, 
~uorometric method [27]. Similarly inhibitory 
levels have been recorded in the mitochondrial 
matrix [28]. 
The presence of carbonic anhydrase in the 
matrix of liver mitochondria has been 
demonstrated [29,30] and a role for this enzyme in 
urea synthesis has been suggested [31]. Inhibition 
of liver mitochondrial carbonic anhydrase by 
acet~olamide reduces citrulline synthesis by more 
than 70% despite the fact that the uncatalyzed rate 
of CO2 hydration is greatly in excess of the rate of 
HCOs- consumption by carbamoyl phosphate syn- 
thetase I (CPS I) at maximum flux. Consequently 
it has been suggested that access of HCOJ- to CPS 
I may be regulated by carbonic anhydrase and the 
two enzymes could possibly be complexed together 
1311. If they do indeed function in some form of 
control unit, it is interesting to note that carbamoyl 
phosphate, whilst inhibiting both enzymes, is 
clearly a more potent inhibitor of carbonic 
anhydrase than of CPS I, for which the icri s lo-19 
mM [32]. 
The possibility that carbonic anhydrase is in- 
volved in control of the activity of carbamoyl 
phosphate synthetase II (CPS II), the cytoplasmic 
enzyme which functions exclusively in pyrimidine 
biosynthesis, seems remote in view of the low 
cytoplasmic levels of carbamoyl phosphate, the 
low activity of CPS II and the tight structural and 
allosteric control under which it is held [33-351. 
However, the likely importance of carbonic 
anhydrase in the regulation of substrate availabili- 
ty for the urea cycle clearly merits deeper 
investigation, 
ACKNOWLEDGEMENTS 
This work was supported in part by grants from 
the Muscular Dystrophy Group, (to N.D.C.) and 
the National Institutes of Health (GM 24681 to 
R.E.T.) 
199 
Volume 165, number 2 FEBS LETTERS January 1984 
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Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate 

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Novel inhibition of carbonic anhydrase isozymes I, II and III by carbamoyl phosphate

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