Semisynthetic enzymes: apoproteins from heme proteins as hydrolases

Proteins having a hydrophobic cavity proximate to which is a residue promoting hydrolysis of functional moieties are effective semisynthetic hydrolases. Heme proteins from which the heme has been removed usually have at least one imidazole residue proximate to the cavity, and thus act as quite effective esterases. Because the size and shape of the cavities of such proteins are capable of broad diversity a wide spectrum of substrates may be hydrolyzed by these semisynthetic enzymes.

BACKGROUND OF THE INVENTION 
It has long been known that enzymes are catalysts par excellence. Not only 
do enzymes vastly increase the reaction rate over that of the uncatalyzed 
counterpart, but enzymes generally show a very high selectivity both in 
the transformation catalyzed and in the substrate acted upon. Thus, a 
relatively small structural change may convert a compound from an enzyme 
substrate, i.e., a material in which a particular transformation is 
catalyzed by an enzyme, to a nonsubstrate, i.e., material in which the 
same transformation is not substantially affected by the same enzyme. The 
high selectivity manifested by enzymes is not limited only to gross 
structural changes in the substrate but also to much more subtle 
differences such as the "handedness," or chirality, of the substrate or 
product being formed. Being chiral molecules themselves, enzymes generally 
readily distinguish between enantiomeric substrates, often showing vast 
differential rate constants for the reaction catalyzed and/or in the 
formation of enantiomeric products. Additionally, enzymes as catalysts 
often are remarkably selective with respect to reaction conditions, such 
as pH, and are effective at or near room temperature. Given these 
attributes, it is understandable that the preparation of synthetic enzymes 
has been a continuing goal of chemists and biochemists. 
However desirable may be the preparation of synthetic enzymes its 
achievement has remained largely a dream rather than a reality. This 
situation undoubtedly arises from the sheer magnitude of the problem. But 
as the mechanisms of enzymatic reactions have been clarified, as the 
structures of enzymes have been elucidated, and as the relation between 
structure, rate, and selectivity has yielded to understanding, the 
nebulous outlines of solutions to the general problem of synthetic enzyme 
preparation have been increasingly better defined. Efforts to prepare 
synthetic enzymes are based on the recognition that enzymatic activity is 
associated with a three-dimensional structure providing a cavity which 
binds a substrate in proximity to a moiety which interacts strongly with a 
particular portion of the substrate. Thus, prior efforts may be classified 
broadly as being directed either toward providing a cavity which strongly 
and/or selectively binds substrates, or placing a reactive moiety in the 
necessary spatial proximity to the substrate site being transformed. 
Exemplifying the former approach is the use of cyclodextrins as 
hydrophobic cavities which extract from aqueous solutions those organic 
molecules having the correct shape. See, e.g., R. Breslow, Science, 218, 
532 (1982). Exemplifying the second approach is the "remote 
functionalization" approach where a template in a molecule controls the 
site of reaction. Idem., ibid. 
Most efforts directed toward constructing enzyme mimics have concentrated 
on hydrolases, and more particularly esterases. Hydrolases may be broadly 
defined as molecules which catalyze the hydrolysis of such functional 
groups as esters, amides, imides, imines, and so forth, whereas esterases 
are a subgroup which catalyze the hydrolysis of esters. Molecules with a 
good binding cavity based on macrocyclic structures and providing high 
rate accelerations have been studied by Cram, J. Am. Chem. Soc., 105, 135 
(1983) and by Breslow, J. Am. Chem. Soc., 103, 154 (1981) as esterases. 
However, the mechanism of ester hydrolysis involves transfer of the acyl 
group from the ester to the esterase, and in the aforementioned studies 
the acyl group remains covalently bound to the esterase precluding further 
catalysis. Stated differently, the esterases of Cram and Breslow remain 
permanently acylated and no turnover is possible. Consequently, such 
esterases are in a real sense not catalysts, since they are permanently 
transformed during the reaction. Polyethylenimines with attached imidazole 
groups have been shown by Klotz, Proc. Natl. Acad. Sci., 68 (2), 263 
(1971) to catalyze ester hydrolysis with turnover for some substrates but 
the rate enhancement and binding capability are only moderate. 
The reaction mechanism of esterases, as representative of hydrolases, can 
be generally depicted as follows. 
##STR1## 
The first stage is a very fast equilibrium binding of the substrate X-Y to 
the enzyme EH, generally through a hydrophobic cavity, characterized by an 
equilibrium constant 1/K.sub.s =k.sub.1 /k.sub.-1, usually called the 
binding constant. Its reciprocal, K.sub.s, is the dissociation constant of 
the bound substrate. The second stage is the transfer of an acyl group of 
the substrate ester to the enzyme with concommitant production of an 
alcohol, XH, in a reaction whose rate constant is k.sub.2. The third and 
last stage is the hydrolysis of the acylated enzyme characterized by the 
rate constant k.sub.3. Usually, k.sub.1,k.sub.-1 &gt;&gt;k.sub.2,k.sub.3. 
Turnover, i.e., regeneration of free enzyme, is determined by the slower 
of k.sub.2,k.sub.3. If k.sub.3 is much less than k.sub.2 then k.sub.3 is 
the rate of turnover, the compound EH is not regenerated in the time frame 
of substrate hydrolysis, and "enzyme" concentration changes during 
hydrolysis, which is a departure from the conventional concept of a 
"catalyst." Conversely, if k.sub.3 is much greater than k.sub.2 (high 
turnover) the compound EH is regenerated far more rapidly than it is 
acylated, and the rate of turnover is determined by k.sub.2. Finally, the 
quantity k.sub.2 /K.sub.M, where K.sub.M =(k.sub.-1 +k.sub.2)/k.sub.1 
=K.sub.s +k.sub.2 /k.sub.1, is usually referred to as the catalytic 
constant. 
Whereas the prior efforts referred to above have afforded systems either 
with (1) low turnover or (2) low binding ability and rate enhancement, the 
claims herein are based on the discovery that a very large class of 
molecules, many readily derivable from naturally occurring proteins, have 
a cavity manifesting good binding propensity with organic substrates and 
which further have active residues which promote or assist hydrolysis, 
especially of esters. Such molecules exhibit high turnover with many 
substrates, and because there is such a large class of such enzyme-like 
molecules (referred to herein as semisynthetic enzymes), there is a good 
probability of finding a semisynthetic enzyme-substrate pair in which the 
system is truly catalytic. One large class of semisynthetic enzymes of 
this invention are heme proteins from which the heme portion has been 
removed. 
SUMMARY OF THE INVENTION 
The purpose of this invention is to provide a method of catalytically 
hydrolyzing organic compounds in aqueous solution using semisynthetic 
enzymes, or hydrolases. In one embodiment the semisynthetic enzyme is a 
heme protein from which the heme has been removed. In a more specific 
embodiment the hydrolase is apomyoglobin. In another embodiment the 
organic compounds are esters. In a still more specific embodiment the 
esters are linear alkyl esters of alkyl, aryl, or aralkyl carboxylic 
acids. Other embodiments will become apparent from the following 
description. 
DESCRIPTION OF THE INVENTION 
The construction of molecules with enzyme-like properties conceptually is 
simple, for such molecules need only (1) bind to the substrate, (2) 
promote the reaction of the bound subtrate, and (3) release the reaction 
products so that the "enzyme" becomes available for binding to additional 
substrate. Those skilled in the art of synthetic enzymes appreciate the 
enormous gulf between the simplicity of this mental construct and the 
enormous complexity and unpredictability of experimental reality. My 
invention is a method of catalyzing the hydrolysis of several types of 
organic compounds using as a catalyst an enzyme-like molecule which is a 
protein having a hydrophobic cavity which selectively binds to the organic 
compound being hydrolyzed and having proximate to the surface of the 
cavity a moiety which promotes or assists hydrolysis of the organic 
compound. An important discovery which makes this invention possible is 
that apoproteins whose parent is a heme protein form a large class of 
hydrolases which are capable of acting on several kinds of substrates, and 
which are especially effective as esterases. The semisynthetic enzymes of 
this invention act as hydrolases, i.e., they catalyze the hydrolysis of a 
variety of functional groups including esters. 
The hydrophobic cavity acts to bind the substrate, preferably more 
selectively than it binds any of the reaction products. Such selectivity 
arises when the reaction products are more polar than the reactants, which 
is usually the case in the hydrolytic reactions being addressed. 
Preferential binding of substrate is also important to minimize product 
inhibition. The size and shape of the cavity will determine what 
substrates may be acted upon, for it should be clear that for a substrate 
to be effectively bound within the cavity there needs to be a good 
geometric fit between the cavity and the substrate. The size and shape of 
the cavity may be readily determined by molecular modeling for proteins 
with a known crystal structure, and a protein with a cavity of appropriate 
size and shape may be chosen with the desired substrate in mind as a way 
of optimizing the catalytic system. 
Proximate to the surface of the hydrophobic cavity there must be at least 
one residue which assists or promotes the hydrolysis being catalyzed. The 
premier example of such a residue is the imidazole group, although serine 
and cysteine residues also are effective in promoting hydrolysis. It is 
necessary that the residue be proximate to the surface of the cavity so 
that it can chemically interact with the substrate which is bound within 
the cavity. Stated differently, if the residue is not proximate to the 
surface of the cavity it will be unable to chemically interact with a 
substrate bound within the cavity, and no catalysis will be possible. 
In general, any protein bearing a prosthetic group which can be removed 
will afford a protein with a hydrophobic cavity, and all such proteins 
containing a residue which promotes hydrolysis may be used in the practice 
of this invention. Heme proteins from which the heme has been removed are 
prime examples of proteins with hydrophobic cavities which are suitable 
for use in this invention, for most heme proteins also have at least one 
imidazole residue at or near the surface of the cavity. The enzymatic or 
non-enzymatic heme protein may be an oxygen carrier, such as hemoglobin 
and myoglobin; an oxidase, as exemplified by catalase and peroxidase 
proteins; and cytochromes of type A(a), B(b) and other specialized 
cytochromes except for the C-type cytochromes. Both prokaryotic and 
eukaryotic cells can be used as sources for heme proteins. Some specific 
examples of heme proteins which may be used in the practice of this 
invention include hemoglobin from fish, mammals or plants (leghemoglobin), 
horseradish peroxidase from horseradish roots, lactoperoxidase from milk, 
chloroperoxidase from caldariomyces fumago, cytochrome C peroxidase from 
yeast, cytochrome b from E. coll, and cytochrome p450 from other bacteria. 
The heme proteins above are only illustrative of those which may be used 
in the practice of this invention, and when used herein "heme protein" is 
a generic term intended to encompass all such materials. Additional 
examples of heme proteins may be found in such references as "The 
Enzymes," J. B. Summer and K. Myrback, Editors, V. II, Part 1, 357-427, 
Academic Press Inc., N.Y. (1951); "Hemes and Hemoproteins," B. Chance, R. 
W. Estabrook, and T. Yonetani, Editors, Academic Press Inc., N.Y. (1966). 
The apoprotein, i.e., a protein without its prosthetic group, is prepared 
by denaturing the protein, removing the prosthetic group, and then 
renaturing the protein. For heme proteins, the native heme may be removed 
by such methods as the acid-acetone method (A. Rossi-Fanelli, E. Antonini, 
and A. Caputo, Biochim. Biophys. Acta, 30, 608 (1958)); D. M. Scholler, 
M-Y R. Wang, and B. M. Hoffman, Methods in Enzymology LII, Part C (1978)) 
or by the acid-butanone method (F. W. J. Teale, Biochim. Biophys. Acta, 
35, 298 (1959)). In both methods the protein is denatured, the heme is 
extracted into the organic phase, and the apoprotein is then renatured in 
aqueous solution. In its renatured form the protein preserves its tertiary 
and quaternary structure including the cavity, but in the absence of the 
heme it has lost its original functional capability. 
The substrates which are used in the practice of this invention are organic 
molecules with at least one hydrolyzable functional moiety. Examples of 
such moieties include esters, thioesters, amides, imidates, and imines. 
Where the catalyst is an apoprotein derived from a heme protein the 
semisynthetic enzyme is a good esterase. Such semisynthetic enzymes also 
may catalyze the hydrolysis of amides, carbamates, imidates, and imines 
but not necessarily with equivalent results. It also needs to be 
emphasized that reference to "esters" also include thioesters, whether it 
is the acyl or alkyl oxygen, or both, which is substituted by sulfur. The 
esters whose hydrolysis may be catalyzed by the semisynthetic enzymes of 
this invention include those of carboxylic acids, sulfonic acids, 
phosphoric acids, phosphorous acids, and the analogous thio acids, where 
carboxylic and thiocarboxylic acid esters are especially desirable 
substrates. 
Using carboxylic esters, A-CO.sub.2 Z, as representative of the substrates 
which may be used, at least one of the carboxyl or alcohol portions must 
be hydrophobic, i.e., either A or Z must be hydrophobic structures. This 
is necessary for the substrate to bind to the hydrophobic cavity. The 
substrate also must be of the size and shape to bind within the cavity. 
For example, where the cavity is long and narrow, linear (unbranched) 
alkyl groups are preferred for either A or Z. So, for example, where the 
semisynthetic enzyme is the apoprotein from sperm whale myoglobin, and 
where the substrates are p-nitrophenyl esters of unbranched alkyl 
carboxylic acids, it appears that a C10 residue is about the maximum that 
can fit into the cavity, and it is found that there is a rate enhancement 
as the alkyl chain is lengthened through C6 with no further enhancement 
observed thereafter. 
On the other hand, where, for example, the cavity is globular a branched 
chain structure for A or Z may be accommodated. In fact, it may be 
necessary to experiment somewhat to optimize the catalyst-substrate 
system, but such experimentation is greatly aided by molecular modeling. 
Clearly, if the results of such modeling indicate that the substrate 
cannot be accommodated within the cavity so as to place the hydrolyzable 
moiety accessible to the reaction promoting residue then either a 
different semisynthetic enzyme needs to be used or the substrate needs to 
be somewhat modified. 
As stated previously, the substrate should be preferentially bound to 
minimize product inhibition. Since the cavity is hydrophobic this implies 
that the product should be more polar than the reactants. Normally this is 
the case in ester hydrolysis, but it is a factor which needs to be kept in 
mind in the practice of this invention. 
For this invention to be practiced the substrate must be water-soluble 
since the reactions are performed in an aqueous system. A water-miscible 
organic solvent may be used, although the amount of such organic solvents 
may need to be limited since they tend to denature the apoproteins which 
act as the semisynthetic enzymes. A common upper limit is 15% by volume, 
although no more than 10% is commonly employed and no more than 5% is 
often preferred. But it needs to be emphasized that the upper limit will 
be determined exclusively by the tendency of the protein to denature. 
Examples of solvents which may be used include the lower aliphatic 
alcohols, such as methanol, ethanol, n-propanol, i-propanol, polyhydric 
alcohols such as ethylene glycol, propylene glycol, 
2-methyl-2,4-pentanediol, glycerol, and so forth, water-soluble ethers 
such as tetrahydrofuran and dioxane, as well as such compounds as 
N-methylacetamide, dimethylformamide, acetonitrile, dimethylsulfoxide, and 
hexamethylphosphoramide. It needs to be understood that such solvents are 
only illustrative of the water-miscible organic solvents that may be used 
in this invention, and such materials are sufficiently well known to one 
skilled in the art that an extensive listing is unnecessary. 
The following description is generally applicable to carrying out this 
invention. However, it cannot be emphasized too strongly that reaction 
conditions are largely determined by the stability of the apoprotein. 
Consequently, although the description here may be applicable to some 
members of the class of semisynthetic enzymes used here the description 
should not be taken too literally. 
Briefly, an aqueous solution of the organic molecule is hydrolyzed, i.e, 
reacted with water, in a solution containing the hydrolase. The 
concentration of the substrate is usually limited by its solubility, but 
it is advantageous to use as high a substrate concentration as is possible 
without encountering serious product inhibition to maximize the amount of 
product formation per unit reaction time. The use of water-miscible 
organic solvents to increase solubility is occasionally employed so long 
as such a solvent system does not denature the semisynthetic enzyme. 
The upper concentration of the apoprotein is limited by its stability, 
i.e., its denaturation rate often increases with increasing concentration. 
For example, a concentration up to about 5.times.10.sup.-4 molar appears 
to be satisfactory where apomyoglobin is used in the practice of this 
invention. A higher or lower concentration may be dictated by apoprotein 
stability under the reaction conditions in the presence of both reactants 
and products. The lower concentration is dictated by the necessity of 
having the semisynthetic enzyme present in an amount effective to catalyze 
the hydrolysis of the substrate. Catalysis may be effective at a 
concentration as low as about 10.sup.-7 molar, although the variables of 
substrate structure, temperature, pH, etc. make a stated lower limit quite 
tenuous. 
The reaction temperature also is dictated by stability of the semisynthetic 
enzyme. When apoprotein derived from, e.g., sperm whale myoglobin is used, 
it often is denatured substantially at a temperature in excess of about 
40.degree. C., and consequently its use is recommended at a temperature 
under about 40.degree. C. However, it needs to be emphasized that even 
where the semisynthetic enzyme is denatured at a higher temperature it can 
be used so long as its denaturation is slower than hydrolysis, although no 
reuse may be possible. 
Finally, the pH at which the hydrolysis is performed also is dictated by 
the denaturation properties of the semisynthetic enzyme. For example, 
where the apoprotein is derived from sperm whale myoglobin a pH range 
between about 6.5 and about 9 is found to cause little if any 
denaturation. 
The following examples merely illustrate this invention and are not 
intended to limit it in any way thereby.

EXAMPLES 
The following descriptions are representative of the methods, techniques, 
and results used in the practice of this invention. Such a description is 
not, and is not intended to be, exclusive and the skilled artisan will 
readily recognize and appreciate the plethora of equivalents which may be 
substituted. 
Preparation of Apomylglobin. The heme group was removed from sperm whale 
metmyoglobin by the acid acetone method of Rossi-Fanelli and coworkers, 
op. cit., from a commercially available sample which was not purified 
prior to use. 
Hydrolysis of p-Nitrophenyl Alkanoates. The experimental methods used in 
the hydrolysis of these esters is exemplified by the hydrolysis of 
p-nitrophenyl caproate. Kinetic runs were performed with the semisynthetic 
enzyme, apomyoglobin, in excess of the substrate. All hydrolyses were done 
in 99.9% aqueous media at 25.degree. C. at pH 8 using 0.05 molar 
tris(hydroxymethyl)aminomethane(TRIS) as a buffer. Each sample cuvette 
containing varying concentrations of apomyoglobin (E.sub.o) in 1 ml of 
0.05M buffer was allowed to equilibrate at 25.degree. C. in a thermostated 
cell holder of a spectrophotometer. To this was added 1 microliter of 
1.13.times.10.sup.-2 M p-nitrophenyl caproate in ethanol and the contents 
thereafter were mixed well. The increase in absorbance at 400 nm arising 
from the p-nitrophenolate ion was monitored as a function of time until 
hydrolysis was complete. The kinetic curves obtained with all enzyme 
concentrations exhibited first order behavior and the rate constants were 
calculated therefrom. Results are summarized in the following Table 1. 
TABLE 1 
______________________________________ 
E.sub.o (10.sup.-5 M) 
k.sub.obs (10.sup.-2 sec.sup.-1) 
______________________________________ 
2.11 1.08 
4.23 1.84 
6.35 2.21 
8.46 2.63 
10.6 2.83 
______________________________________ 
When the observed rate constants, k.sub.obs were plotted according to the 
Lineweaver Burk formulation, 
##EQU1## 
a linear relationship was obtained with K.sub.M =7.4.times.10.sup.-5 M and 
k.sub.2 =4.93.times.10.sup.-2 sec.sup.-1. In the absence of apomyoglobin 
the hydrolysis of a solution of 1.13.times.10.sup.-5 M of the same ester 
under the same conditions proceeded with the rate constant of 
2.7.times.10.sup.-5 sec.sup.-1. The enhancement achieved with the 
semisynthetic enzyme is therefore 1800. 
Turnover was demonstrated by comparing the rate of hydrolysis of a 
1.times.10.sup.-5 M solution of p-nitrophenyl acetate by 5.times.10.sup.-5 
M apomyoglobin to the hydrolysis rate of 1.times.10.sup.-5 M ester in the 
presence of a mixture of 5.times.10.sup.-5 M apomyoglobin and 
5.times.10.sup.-5 M ester which was first allowed to react to 95% 
completion. Both rates were identical, verifying that in this case 
acylation rather than deacylation is the rate determining step, or k.sub.3 
is much greater than k.sub.2. Where the same type of experiment yielded a 
decrease in catalysis rate in those instances the deacylation may become 
rate limiting. Some results of ester hydrolysis catalyzed by apomyoglobin 
are summarized in Table 2, where k.sub.2 /k.sub.M is often referred to as 
the "catalytic constant," and k.sub.un is the hydrolytic rate constant in 
the absence of apomyoglobin. 
__________________________________________________________________________ 
ESTER HYDROLYSIS BY APOMYOGLOBIN 
p-Nitrophenyl 
Ester K.sub.2 (sec.sup.-1) 
K.sub.M (M) 
k.sub.2 /K.sub.M (M.sup.-1 sec.sup.-1) 
k.sub.2 /K.sub.un 
Turnover 
__________________________________________________________________________ 
Acetate 
5.8 .times. 10.sup.-3 
4.3 .times. 10.sup.-4 
13.5 134 
k.sub.3 &gt; k.sub.2 
Propionate 
1.6 .times. 10 -3 
2.5 .times. 10.sup.-4 
6.4 42 k.sub.3 &gt; k.sub.2 
Butyrate 
3.8 .times. 10.sup.-3 
1.43 .times. 10.sup.-4 
26.4 127 
k.sub.3 .ltoreq. k.sub.2 
Caproate 
4.93 .times. 10.sup.-2 
7.4 .times. 10.sup.-5 
67 1800 
k.sub.3 &lt; k.sub.2 
Caprate 
3.0 .times. 10.sup.-2 
6.1 .times. 10.sup.-5 
49 4600 
k.sub.3 &lt; k.sub.2 
__________________________________________________________________________ 
To ascertain that our remarkable catalytic rates result from the active 
site only of the empty heme pocket or cavity the following experiments 
were performed. In the first experiment reconstituted myoglobin was 
prepared from apomyoglobin and hemin, and the hydrolysis of p-nitrophenyl 
caproate was determined as previously described. It was found that the 
hydrolysis rate was not more than twice that of the spontaneous 
(uncatalyzed) rate. This eliminates the possibility that periperal lysine 
and histidine residues catalyze hydrolysis. In the second experiment the 
rate of hydrolysis of a solution of p-nitrophenyl acetate in buffer 
containing apomyoglobin, prepared as described above, was compared with 
the rate of hydrolysis in a similar solution which was also 8 molar in 
urea to ensure denaturation of apomyoglobin. After 150 seconds the 
reaction was 98% complete in the first solution, but only 2% complete in 
the urea-containing solution. This shows that the denatured protein is 
catalytically ineffective. These results establish the heme cavity as the 
active site of the semisynthetic esterase.