81Br-nmr studies of carbonic anhydrase

BIOCHEMICAL

Vol. 51, No. 2, 1973

8$r-NMR

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STUDIE S 0 F CARBONIC ANHYDRASE*

Raymond L. Ward Livermore Laboratory, University Livermore, California 94550

Lawrence

of California

and Philip L. Whitney Department of Biochemistry University of Miami School of Medicine Miami, Florida 33152 Received

January

24,

1973

Summary have been made on high (bovine; BCA) and low 8b r nmr measurements (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethygi CAM-HCAB). The high specific activity form of the enzyme, BCA, e_xhibits a Br line broadening which is determined by the lifetime of Br bound to the zinc ion of the enzyme, The low specific activity form of the enzyme, HCAB under similar conditions of concentration, pH, etc., does not exhibit a BlBr nmr line broadening. Cl-/Brcompetitive binding studies, using 35Cl nmr, is due to an increase suggests that the failure to observe 81Br broadening in the lifetime of a zinc bound Br-. An increase in this lifetime by a factor of lo-100 over that exhibited by BCA is sufficient to abolish the li e broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a 8 1Br nmr line broadening. Estimates of the lifetime of zinc bound Br-, r , are 4 x 10-7 sec. for CAM-#CAB at pH 8 and 1 x 10-7 set for BCA at pH 7. & e lifetime for Br- bound to HCAB is estimated to be >lO- 6 sec. Chloride

nmr probe

have shown that

Cl-

can bind

Furthermore,

differences

environments

at the

of the B (@X6)

enzyme.

active

directly

sites

For example,

the pKh for

the

zinc

8.7 and 7.0

respectively

of the various

Cl-

study

in

zinc

binding

the two isoenzymes,

of the

reflect

and low

studies

activity

of carbonic

ion

data

of the high

low and high ion

forms

to the

in 35Cl relaxation

and C (EAC),

A temperature

studies

anhydrase

enzyme

differences specific

in

activity

of human carbonic

forms

of the enzyme,

extrapolated

(1,2).

forms anhydrase

reveal

to zero

that

Cl-,

is

(3). of the

Cl-

line

broadening

"This work was performed in part under the auspices Energy Commission and by grant GB-26444 to P.L.W. Foundation

Copyright 0 1973 by Academic Press, Inc. AI1 rights of‘ reproduction in any form reserved.

343

in the presence

of XXB

of the U.S. Atomic from the National Science

Vol. 51, No. 2, 1973

BIOCHEMICAL

200

,

b I

-

I,

I

AND BlOPHYSlCAL

I

I

I,,

Solid - Bovine

.

,

,

I,

RESEARCH COMMUNICATIONS

,,,I

CA

Open - Human CAB D

4loo- & *.

A

. : j,,, ,,,yJy.

.

;

.

50

0 7

a

9

10

11

PH

Fig. 1 Net line width, dune,, i.e.,

Acbsd - Au,,-,

vs. pH for solutions

of bovine carbonic anhydrase (solid symbols), 0.8 mg/ml, and human carbonic anbydrase B (open symbols), 4 mg/ml, in 0.5 M NaBr. The pKa for the bovine enzyme is 8.6.

indicated

that the broadening is determined by the lifetime

the zinc ion of the enzyme (3).

Further comparisons af line broadening using

both 35Cl and 37Cl isotopes also indicates dominating factor

of Cl- bound to

that the exchange rate is the

in the line broadening (4).

8$ r nmr studies of CA should complement the Cl- studies. viously (3).

We have pre-

shownthat Br- and Cl- compete for the Zn site of the bovine enzyme Data in the literature

indicate

that although the 8$r

1'ine width

increases in the presence of HCAD, there is no change in the broadening upon the addition of a sulfonamide inhibitor in conflict

(5).

These observations appear to be

with the Cl- studies (1,2).

In examining this point further,

we observe that 8$r nmr line broaden-

ings in the presence of BCA do complement our Cl- results. broadening similar

The '$r-

line

observed is pH dependent (Figure 1) and this pH behavior is quite

to that of Cl- binding observed with 35 Cl nmr. Furthermore, the '$r-

line broadening is reduced in the presence of cyanide and acetazolamide, a powerful sulfonamide inhibitor

(4).

Titrations

344

with these inhibitors

yield

Vol. 51, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

PH

Fig.

2

Net line

width,

nvnet,

i.e.,

modified (carboxyamidomethyl) NaBr. The pKa is 9.6.

results

identical

equivalent Since

the

the exchange

rate

would

the fast

moments.

We observe therefore

for

be 1.39; a ratio

the

under

also

shown in Figure

this

more concentrated

pertained,

line

identical

solution

reasonable were

the bovine broadening

square

line

of the

system. for

five

nondescript

line that

line

width be

widths quadrupole

The rate

of Br-

BCA.

The line times

pH.

of CA, would

of concentration,

must be ascribed 345

in the

of the

appreciably.

this

at high

the case the

M

of one

to expect

same solution

of the

conditions

At present

observed

of

in 0.5

addition

factor

the ratio

in the ratio

the

the

to that

this

a solution

B, 3.4 mg/ml,

i.e.,

is

in the

of 1.0 for

pH for

determining

If

of HCAH containing 1.

the

to Br-.

a$ r nmr linewidth

a solution

width

of BCA, it

limit

determines

HCAB, however,

observed

is

, measured

exchange

in Hz would

does not affect

apply

isotopes

measured

exchange

of Cl-

in the presence

of 79Br/8$r If

the line

vs.

anhydrase

by 35 Cl nmr,

obtained

reduces

same mechanism

ratios 1.0.

to those

of Cl-

- AuBr-,

human carbonic

of inhibitor

broadenings

nzbsd

pH, etc. broadening

the amount broadening

to a variety

of BCA is observed

of effects

in

Vol. 51, No. 2,1973

including

BIOCHEMICAL

viscosity,

nonspecific

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

halogen binding,

of broadening due to the zinc ion.

Examination of 35Cl line broadenings

produced by HCABin the presence of varying indicates

and perhaps a small amount

amounts of Br-,

however,

competition between Cl- and Br- for the zinc site.

Wethus suggest

that for HCABthe exchange rate of zinc bound Br-

is sufficiently

no effect

A decrease in the rate of

is observed on the free Br- line width.

exchange by a factor would be sufficient

slow that

of lo-100 over that estimated for the bovine enzyme to explain our results.

We have also examined the carboxamidomethyl form of the humanB enzyme, cm-HCAB. This derivative

of HCABis prepared by the reaction

mide with a single histidyl

activity yield

residue (His 204) located near the active

It possesses 3% of the CO2 hydrating

(6).

of the native enzyme (7). information

of iodoaceta-

quite similar

activity

site

and 30s of the esterase

Chloride nmr studies of this derivative

to that reported for the native enzyme (4).

In contrast to the 8%r studies of the native enzyme, HCAB, however, the modified enzyme exhibits

a 8?Brb roa aening quite similar The modification

the bovine enzyme (Figure 2). carboxysmidomethyl

group thus results

to that observed for

of His-204 with an uncharged

in a decrease in the lifetime

of bro-

mide ion bound to the zinc ion. The discussion presented elsewhere (3) for 35Cl broadening in the presence of BCA can be extended to include the Br- results. between line width and exchange lifetime rM is the exchange lifetime

The relationship

is given by 0~ = f/n(Ta

+ TM) where

of bound bromide,

tion time, and f is the fraction

of total

is the transverse relaxaTal bromide which is bound to the zinc

ion of carbonic anhydrase. The slow-exchange limit, presented here and the lifetime with a knowledge of f.

i.e.,

TM >> Ta,

of zinc bound bromide ion can be estimated

The fraction

of bound bromide, f,

from the value of KI obtained from chloride(4).

pertains to the bromide results

can be calculated

bromide competitive

experiments

These values are 32 mMfor BCA and 32 mMfor CAM-HCABat pH 7.5 and 16

346

Vol. 51, No. 2, 1973

BIOCHEMICAL

Hc.AB at pH 8.

mu for

values

to %

this

small.

and 1 x 10 -7 sec. correspond

The f values

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are,

Calculated

at pH 7 for

values

of magnitude

less

than

that

(8).

Aqueous

ions

are,

however,

anhydrase

is

of Br-

in a slightly bound

observed

particularly

times

for

aqueous

zinc

measurement these

as a function

(9).

two possibilities

the

dissociative

vs.

[Br-]

is

agreement

mechanism obtained

with

These ion

of total

studies

environment

be different

of the

for

HCAB are

also

where

zinc

slow

additional high

support

and low activity

perturbed

bromide

ion.

Other

by the modification

width

cobalt the

result

properties

the

anhydrase

residue

is reflected

is

(10).

zinc must

- 204 in

in a decrease of the active

are

in

enzyme that

of carbonic

that

of AU-1 net

substituted conclusion

and these

between

plot

This

of histidine group

i.e.,

A line

line

limit.

forms

modification

carboxyamidomethyl bound

for

sec.

enzyme indicate

a straight

of the

The life-

can distinguish

exchange

carbonic

discussed

site

in of

else-

(7).

Materials

and Methods

A carboxamidometbyl (CAM-HCAB)

was prepared

measurements frequency 79Br

i.e.

in

(6). -b

+ Br-.

on the bovine

nmr studies

Furthermore,

HCAB by an uncharged lifetime

BCA in the

anion

yield

(2).

Measurements

zinc

solutions

or dissociative,

concentration

is operative,

for

similar

Br-

an

bromide

the

to be > 10

exchange mechanism is either competitive -Y -3c + Br p E-Zn-Br + Br- or E-Zn-Br2 E-Zn

pH values

approximately

environment

estimated

at pH8

These

whereas

tetrahedral

in HCAB is

are

The BrE-Zn-Br-

sensitive

4 x 10 -7 sec.

rM are

octahedral

distorted

to zinc

for

These

to the maximum broadenings.

zinc

not

CAM-HCAB and BCA respectively.

order

time

however,

were swept

at 15.036

measurements,

derivative and purified

carried

out

spectrometer.

carried

by affinity

at a fixed The 8$r

field

out

at

32°C.

347

carbonic

value

anhydrase

(7).

chromatography

measurements

at 5.878 MHz. All

MHz, and 35Cl were

of human erythocyte

of 14,098

B

All

gauss with

a

were made at 12.206 MHZ,

titrations,

including

pH

Vol. 51, No. 2,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References Ward, Ward, Ward, Ward,

R. R. R. R.

L., L., L. L.,

Biochemistry 3 1879 (1969). Biochemistry 3 2447 (1970). and Cull, M. D., Arch. Biochem. Biopmsics 150, 436 (1972). to be published. Forsen S., and Lindqvist, I. Biochem.

E~~~YY&~'6~~~Bi~~

(19693.

Lindskog, S., Henderson, L. E., Kannan, K. K., Liljas, A., IQ-man, P. O., and Strandberg, B. in The Enzymes (Bayer, P. D., ed) 3rd Ed., Vol. 5, pp 587-665, Academic Press, NewYork. Whitney, P, L., J, Biol. Chem., accepted for publication. Eigen, M., and Wilkins, R. G., Mechanism of Inorganic Reactions, Advances in Chemistry Series, No. 49, (1965), Washington, D.C., American Chemical Society. Navon, G., Shulman, R. G. Wyluda, B. J., and Yamane, T., J. Mol. Biol. 51.

15 (1970).

Taylor,

P. W., Feeney, J., and Burgen, A.S.V.,

0971).

348

Biochemistry 10, 3866