Catalytic Antibodies (Abzymes) and Enzyme Dynamics

If enzymes accelerate reactions by preferentially binding to and stabilizing the transition state, might an antibody raised against a suitable transition state analog be catalytically active?
Desirable features of antibodies: 1. easily prepared in great diversity 2. very stable 3. structurally well-characterized

Schematic structure of an antibody (IgG):

VH S S VL S S S S CL CH 1 S S S S C H2 S S CH 2 S S S S S S C H1 S S CL S S S S VL VH S S

Light and Heavy subunits, Variable and Conserved domains within each subunit.

S CH 3 S

S C H3 S

CHx - conserved domain, heavy chain VL - variable domain, light chain

Some definitions: antigen - a substance that elicits antibody or immune cell formation/proliferation hapten - a small substituent on a protein that can elicit an immune response monoclonal antibody - an antibody obtained from a cultured immune cell
population derived from a single activated B lymphocyte

polyclonal antibody - the antibody fraction isolated from the plasma of an

animal treated with an antigen General protocol:
synthesize analog covalently link analog to carrier protein innoculate animal isolate and clone lymphocytes analyze MAb's

Some reactions catalyzed by monoclonal antibodies prepared to date: de-esterification (hydrolysis)

O R C + H2O O-R' RCO2H + R'OH R

transesterification
O C + R"OH R O-R' R'OH O + C O-R"

cis-trans isomerization
R R R' R' X HC R R' CH H

-elimination
H C R + HX C H R'

Diels-Alder reaction
O

Claisen rearrangement
O R
H

OH R R

Ester hydrolysis by catalytic antibodies (Tramantano et al. (1986) Science 2 3 4 , 1566-1570

Pollack et al. (1986) Science 2 3 4 , 1570-1573)

Reaction mechanism: H F3C N O O C H HON CH3 O

OR HO OR'

H F3C N O O P O O H N O CH3
2

O C

+ HO

H N CH3 O

H N F3C O

Structure of the transition state analog

Catalytic properties of the monoclonal antibody: kcat = 0.03 s-1 1/v (sec/nM) Km = 1.9 M kcat/Km = 1.5 x 104 M-1s-1 Ki = 0.16 M kcat/kuncat = 960 30
o

[analog]
x

20
o x o ox xo o o o

10

2 1/[S] (M-1)

Judicious placement of a charged group complementary to that desired in the antibody can greatly facilitate catalytic activity:
1274-1275

Janda et al. (1990) J. Am. Chem. Soc. 1 1 2 ,

O O N H O O O-

HO O O
-

HO R

inhibitor (amide of acetate) haptens used: N OH vs.

+

O N H hapten

O O N O

N R CH 3 Mab kcat 5 x 10-3 min-1 Km 1.1 mM kcat/kuncat Ki 6 ~ 10 83 M

Hydrolysis of amide (peptide) bonds by a catalytic antibody has also been shown: kcat/kuncat = 2.5 x 105
Janda et al. (1988) Science 2 4 1 , 1188-1191
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Proximity effects in antibody-catalyzed reactions (antibodies as "entropic traps"):

Diels-Alder reaction O HN O OO diene O + N O CH3 NH O dienophile
-

O N O O O NH O

CH3 NH

transition state O
-

O R N O

analog O
-

O N R NH H O O

O
x

H NH O

30 25
x

25
o

1/v (sec)

1/v (sec)

20
x

x o

o o x x

20 15 10 5 o
o o x ox o xo o o

kcat = 0.67 s-1 Kmdiene = 1.13 mM Kmdienophile = 0.74 mM [analog] kcat/Kmdiene = 580 M-1s-1 kcat/Kmdienophile = 900 M-1s-1

15 10 5
x x o o x x o o

x x
o

x o x o

o x x o o

[dienophile]
o

x x
o o o

1/[diene] (M-1) Claisen rearrangement

CO 2-

1/[diene] (M-1)

kuncat = 2.0 M-1s-1

O 2C O CO 2-

CO 2-

O 2C

O OH

CO 2OH OH

-O2C

10

o kcat = 0.87 sec-1 x o x o

10 20

1/v (sec)

O OR inhibitor: R = H O hapten: R = N H

CO2

8 6 4

o ox 2 ox o xo o

[hapten] o

Km = 480 M Ki = 0.15 M kcat/kuncat ~ 104

N 2+

1/[substrate] (mM-1)
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Ironically, however, even "off-the-shelf" proteins can exhibit gratuitous enzyme activity
Hollfelder et al. (1996) Nature 3 8 3 , 60-63 versus Thorn et al. (1995) Nature 3 7 3 , 228-230.

O 2N

H N O

:B

The transition state and hapten used in the CAb study: B

bovine serum CAb albumin O 2N OH N

O 2N

H N O

H3C H3C

H N N NH2 CO 2H

catalytic antibody kcat = 40 min-1 Km = 120 mM kcat/Km =3.3 x 105 M-1min-1 kacetate = 8.4 x 10-3 M-1min-1 bovine serum albumin (pH 9)
water

kcat/kuncat was reported to be ~4 x 107 !!

But:

kcat = 30 min-1 Km = 700 mM kcat/Km = 4.2 x 104 M-1min-1

water kacetate = 8.4 x 10-3 M-1min-1

3 kacetate = 1.7 x 105 M-1min-1

CH CN

The degree of catalysis is comparable to that observed for a catalytic antibody raised against a transition state analog. It turns out that BSA has a hydrophobic pocket at which the substrate binds, and and there is a specific lysine residue found at the bottom of this hydrophobic pocket. The predominant effect appears to be one of desolvation of the substrate on binding to serum albumin.

kcat/Km (x 10-4 M-1s-1)

pKa = 8.95

10

12

pH
5

A detailed analysis of whats going on in the course of the affinity maturation process that leads to generation of a catalytic antibody indicates that things are quite a bit more complicated than had initially been expected. Occasionally, affinity maturation results in a catalytic antibody that is actually less effective as a catalyst than the immature germ-line antibody. It is thus clear that there is not always a direct correlation between affinity for the hapten and catalytic power.
(Patten et al. (1996) Science 2 7 1 , 1086-1091 Ulrich et al. (1996) Nature 3 8 9 , 271-275)

How this might happen:

Hilvert (2000) Annu. Rev. Biochem. 69, 751-793.

Differential effects on kon and k off in the course of antibody maturation - The greater affinity for hapten that arises as a result of affinity maturation of the antibody is due for the most part to a decrease in koff rather than an increase in kon. Thisis bad from a catalytic standpoint since things tend to get stuck in the active site. Non-additive effects of mutations in the course of maturation Mutations in the VL and V H fragments tend to have additive effects on the catalytic properties of the CAb, but the effects of individual mutations within one or the other chain are not always additive maturation involves an "either/or" process, not "both". Loss of protein flexibility in the course of antibody maturation The specific mutations that take place in the germ-line antibody during affinity maturation are not always at the hapten binding site and can involve changes in conformation and/or flexibility of the mature antibody rather than changes in the hapten binding site. To the extent that maturation reduces flexibility in the active site, catalysis may be compromised because there changes in shape must occur as substrate becomes product.

Enzyme dynamics and function

Protein dynamics play out in the function of enzymes in a variety of ways: Dynamics of substrate binding in the active site Changes in domain-domain interactions to create the active site Reorientation of amino acid residues in the active site Breathing of the protein backbone and local motion of side-chains to create greater access to the active site. CO binding to myoglobin Braunstein et al. (1988) Proc. Natl. Acad. Sci. USA 85, 8497.
diffusion through solvation shell diffusion into heme pocket diffusion to heme formation of Fe-CO bond solvation shell

free energy

Fe

CO trapped in heme pocket CO in protein matrix

free CO

"docked" CO

bound MbCO

reaction coordinate

There are two basic of experimental approaches:

1. Pulse-probe methods, relying on, e.g., photoexcitation of the sample 2. magnetic resonance methods a. dynamic phenomena that influence lineshape

slow
H HO H H H R

intermediate

fast

With tyrosine residues, for example, immobilized residues give four aromatic proton resonances, while residues freely rotating about the C bond give two (as the two H and H protons become equivalent).
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b. time-averaged phenomena that can be treated statistically

The timescale of various protein motions

vibrations of individual chemical bond - ~10 fsec rotation of amino acid sidechains - 1-100 psec motions of short loops and bends - nsec to sec reorientation of domains with respect to one another - ~1 msec quaternary conformational changes - ~1 msec protein breathing - seconds to days

Consider Cyclophilin A Eisenmesser et al. (2002)

N H O N

Science 2 9 5 , 1520-1523.

ktranscis

Kdcis

E
Kd

trans

EStrans
kcistrans

EScis
O N

H N O

Some C chemical shifts are insensitive to biinding of substrate, others are:

The transverse relaxation rate R2 is sensitive to motions on the sec-msec timescale, those on which substrate binding and isomerization occur. Only a few residues show increases in R2 on binding substrate, reflecting the interconversion of E, EScis and EStrans states. Arg 55 is known from the crystal structure of the ES complex to interact with the peptide bond to the C-terminal side of the substrate proline residue.

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Most residues that are affected by substrate binding behave like Lys 82, with a clear maximum value for R2 at intermediate concentrations of substrate, when all three enzyme states are populated. This is the expected behavior for effects on R2 involving simply substrate binding. For Arg 55, on the other hand, R2 plateaus and does not significantly diminish at high [S], the type of behavior expected when the effect on R2 is due to isomerization rather than substrate binding.

Fits to the profiles using known values for the rates of substrate binding and dissociation yield values for kcis-trans and ktrans-cis of 5,000 s-1 and 9,000 s -1, respectively, in excellent agreement with the experimentally observed rates of catalysis from conventional assays. Motion of Arg 55 in the ES complex and catalysis are strongly correlated.

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Although a crystal structure is available only for CypA with bound substrate in the cis conformation, there are several residues from 98 to 104 that have increased R2 values upon binding of substrate this suggests a role in binding substrate. A model has been proposed in which rotation about the prolyl peptide bond results in the C-terminal portion of substrate swinging down into contact with a -strand that includes this portion of the protein sequence.

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Ultimately, the importance of protein dynamics on enzyme kinetics has to do with its effect on the potential energy surface and reaction coordinate diagram of the system. It is to be expected that some protein configurations, even those rather far from the equilibrium configuration, will have significantly lower activation energies than others. The point is that the activation barrier is not static but fluctuates due to motions ranging from bond vibrations (~10-14 s time-scale) to large-scale domain-domain motions (~10-3 sec time-scale).

Potential Energy

less effective catalysis more effective catalysis

P
Reaction Coordinate

One can imagine an unfavorable protein configuration that is populated only 0.1% of the time, but which has a substantially lower activation barrier to reaction. If the activation barrier were as much as 10 kcal/mol lower than that for the average configuration, it would amount to a 106-fold greater rate acceleration in the unfavorable configuration. The result would be that almost all the catalytic throughput (1,000 to 1) would take place in the thermodynamically unfavorable but catalytically more effective configuration. This should scare the hell out of protein crystallographers!

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Also, bear in mind that the reaction coordinate is just one pathway on a potential energy surface that describes the full system. It represents the lowest-energy path from reactants to products, and the pathway itself may well change (usually only modestly) with fluctuations in the potential energy surface as a result of changes in protein conformation.

Since the entire potential energy surface fluctuates as a result of changes in protein configuraton, the energy barriers along specific pathways connecting specific points will fluctuate, as will the lowest-energy pathway from one to another.

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Protein dynamics are important even in those situations where tunneling is thought to occur (e.g., alcohol dehydrogenase).
Kohen et al. (1999) Nature 3 9 9 , 496-499. Kohen and Klinman (2000) J. Am. Chem. Soc. 1 2 2 , 10738-10739.

Zn

2+

O R

HH

H CONH2

Zn2+

O R

H CONH2

alcohol

N+ R

NAD

aldehyde

N R

NADH

The temperature-dependence of both primary and secondary isotope effects strongly suggest that the reaction proceeds by tunneling through rather than traversing the transition state. Not only this, but tunneling is coupled to vibrations both in substrate and in side chains of the polypeptide.

potential energy

reaction coordinate
Consider the C-O bond stretch:
maximum C-O distance most likely hydride transfer to C=O least likely hydride transfer from C-OH mean C-O distance minimum C-O distance most likely hydride transfer from C=O least likely hydride transfer to C-OH

O O O
oxygen displacement

C C C

The idea is that side chains influence reaction rate by banging off the substrates, influencing their vibrations and relative distances. Val 203, opposite NAD(H) from the alcohol, has been specifically implicated from molecular dynamics calculations. This type of effect may be particularly important in enzymes from thermophilic organisms, accounting in part for their unusually low activities at ambient temperatures.
Antoniou and Schwartz (2001) J. Phys. Chem. B 1 0 5 , 5553-5558.
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