Chemically modified enzymes

Details Chemically modified enzymes Chemically modified enzymes

1997-11-25

2001-08-21

Witz, Jean C. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S221000, C435S069100

Reexamination Certificate

active

06277617

ABSTRACT:

BACKGROUND OF THE INVENTION
Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish, V. W. et al. (1995)
Angew. Chem.,
Int. Ed. Engl. 34:621). However, the latter procedures are not generally easy to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.
Changing enzyme properties by chemical modification has been explored previously, with the first report being in 1966 by the groups of Bender (Polgar, L. et al. (1966) J. Am. Chem. Soc. 88:3153) and Koshland (Neet, K. E. et al. (1966)
Proc. Natl. Acad. Sci. USA
56:1606), who created a thiolsubtilisin by chemical transformation (CH
2
OH→CH
2
SH) of the active site serine residue of subtilisin BPN' to cysteine. Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by Wu, Z. -P. et al. (1989)
J. Am. Chem. Soc.
111:4514; Bell, I. M. et al. (1993)
Biochemistry
32:3754 and Peterson, E. B. et al. (1995)
Biochemistry
34:6616, and more recently by Suckling, C. J. et al. (1993)
Bioorg. Med. Chem. Lett.
3:531.
Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural, wild-type, enzymes can never hope to accept all structures of synthetic chemical interest, nor always to transform them stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in to altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigued chemists for many years, and continues to do so. The inventors have adopted the combined site-directed mutagenesis-chemical modification strategy since it offers virtually unlimited possibilities for creating new structural environments at any amino acid location.
U.S. Pat. No. 5,208,158 describes chemically modified detergent enzymes wherein one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with a C
1-6
alkyl.
Although U.S. Pat. No. 5,208,158 has described altering the oxidative stability of an enzyme, it would also be desirable to develop one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile and surface binding properties for use in, for example, detergents or organic synthesis.
SUMMARY OF THE INVENTION
There exists a need for enzymes such as proteases that have altered properties. As such, the present invention provides modified enzymes that have one or more amino acid residues replaced by cysteine residues. The cysteine residues are modified by replacing the thiol hydrogen with a substituent group providing a thiol side chain selected from the group consisting of:
a) —SR
1
R
2
, wherein R
1
is an alkyl and R
2
is a charged or polar moiety;
b) —SR
3
, wherein R
3
is a substituted or unsubstituted phenyl;
c) —SR
4
, wherein R
4
is substituted or unsubstituted cyclohexyl; and
d) —SR
5
, wherein R
5
is C
10
-C
15
alkyl.
In preferred embodiments, the thiol side chain groups —SR
3
and —SR
4
above, further comprise an alkyl group, R, which is placed before either R
3
or R
4
to form —SRR
3
or —SRR
4
. R is preferably a C
1-10
alkyl.
With regard to the thiol side chain group —SR
1
R
2
, R
2
can be positively or negatively charged. Preferably, R
2
is SO
3
−
, COO
−
or NH
3
+
. Further, R
1
is preferably a C
1-10
alkyl.
Preferably, the enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Also, preferably, the amino acids therein replaced by cysteines are selected from the group consisting of asparagine, leucine, methionine or serine. More preferably, the amino acid to be replaced is located in a subsite of the protease, preferably, the S
1
, S
1
′ or S
2
subsites. Most preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166 where the numbered position corresponds to naturally-occurring subtilisin from
Bacillus amyloliquefaciens
or to equivalent amino acid residues in other subtilisins, such as
Bacillus lentus
subtilisin.
In a particularly preferred embodiment, the enzyme is a
Bacillus lentus
subtilisin. In the most preferred embodiments, the amino acid to be replaced by cysteine is N62 and the thiol side chain group is selected from the group:
—S
1
R
2
wherein R
1
is CH
2
and R
2
is CH
2
SO
3
−
;
—SRR
3
wherein R is CH
2
and R
3
is C
6
H
5
;
—SRR
4
wherein R is CH
2
and R
4
is c-C
6
H
11
;
—SR
5
wherein R
5
is n-C
10
H
21
; or
the amino acid to be replaced by cysteine is L217 and the thiol side chain group is
—SR
5
wherein R
5
is n-C
10
H
21
.
The present invention further provides modified enzymes that have one or more amino acid residues replaced by cysteine residues. The cysteine residues are modified by replacing the thiol hydrogen with a substituent group providing a thiol side chain —SR
6
wherein R
6
is a C
1-6
alkyl and the amino acid residues to be replaced by cysteine are selected from the group consisting of asparagine, leucine, and serine. Preferably, the enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Most preferably, the amino acid is located in a subsite of the protease, preferably, the S
1
, S
1
′ or S
2
subsites. Most preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166. Preferably, the enzyme is a
B. lentus
subtilisin, the amino acid to be replaced by a cysteine is N62 or L217 and the thiol side chain group is —SR
6
wherein R
6
is CH
2
C(CH
3
)
3
or C
5
H
11
.
The present invention provides a method of producing a modified enzyme, including providing an enzyme wherein one or more amino acids have been replaced with cysteine residues and replacing the thiol hydrogen of the cysteine residue with a subtituent group providing a thiol side chain selected from the group consisting of:
a) —SR
1
R
2
, wherein R
1
is an alkyl and R
2
is a charged or polar moiety;
b) —SR
3
, wherein R
3
is a substituted or unsubstituted phenyl;
c) —SR
4
, wherein R
4
is substituted or unsubstituted cyclohexyl; and
d) —SR
5
, wherein R
5
is C
10
-C
15
alkyl.
In preferred embodiments, the thiol side chain groups —SR
3
and —SR
4
above, further comprise an alkyl group, R, which is placed before either R
3
or R
4
to form —SRR
3
or —SRR
4
. R is preferably a C
1-10
alkyl.
With regard to the thiol side chain group —SR
1
R
2
, R
2
can be positively or negatively charged. Preferably, R
2
is SO
3
−
, COO
−
or NH
3
+
. Further, R
1
is preferably a C
1-10
alkyl.
Preferably, the enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Also, preferably, the amino acids therein replaced by cysteines are selected from the group consisting of asparagine, leucine, methionine or serine. More preferably, the amino acid to be replaced is located in a subsite of the protease, preferably, the S
1
, S
1
′ or S
2
subsites. Most preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166 where the numbered position corresponds to naturally-occurring subtilisin from

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