Chemically modified mutant enzymes and methods for producing...

Details Chemically modified mutant enzymes and methods for producing... Chemically modified mutant enzymes and methods for producing...

1999-01-21

2001-09-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S019000, C435S183000, C435S263000, C435S113000, C435S440000, C435S124000, C435S219000, C435S220000, C435S222000, C424S094640, C426S053000, C510S220000, C510S238000, C510S236000, C510S320000, C510S392000, C510S530000

Reexamination Certificate

active

06284512

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to chemically modified mutant enzymes and a method of producing them and a method of screening them for amidase and/or esterase activity.
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 et al.,
Angew. Chem
., Int. Ed. Engl., 34:621-633 (1995)). However, the latter procedures are difficult 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 et al.,
J. Am. Chem. Soc
., 88:3153-3514 (1966)) and Koshland (Neet et al.,
Proc. Natl. Acad. Sci. USA
, 56:1606-1611 (1966)), 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 (Wu et al.,
J. Am. Chem. Soc
., 111:4514-4515 (1989); Bell et al.,
Biochemistry
, 32:3754-3762 (1993)) and Peterson (Peterson et al.,
Biochemistry,
34:6616-6620 (1995)), and, more recently, Suckling (Suckling et al.,
Bioorg. Med. Chem. Lett
., 3:531-534 (1993)).
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 be transformed 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 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.
U.S. Pat. No. 5,208,158 to Bech et al. (“Bech”) 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 C
1-6
alkyl.
Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to identify 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. In particular, enzymes, such as subtilisins, tailored for peptide synthesis would be desirable. Enzymes useful for peptide synthesis have high esterase and low amidase activities. Generally, subtilisins do not meet these requirements and the improvement of the esterase to amidase selectivities of subtilisins would be desirable as would a rapid identification of subtilisins with improved esterase to amidase selectivities. However, previous attempts to tailor enzymes for peptide synthesis by lowering amidase activity have generally resulted in dramatic decreases in both esterase and amidase activities. Previous strategies for lowering the amidase activity include the use of water-miscible organic solvents (Barbas et al.,
J. Am. Chem. Soc
., 110:5162-5166 (1988); Wong et al.,
J. Am. Chem. Soc.,
112:945-953 (1990); and Sears et al.,
Biotechnol. Prog.,
12:423-433 (1996)) and site-directed mutagenesis (Abrahamsen et al.,
Biochemistry,
30:4151-4159 (1991); Bonneau et al.,
J. Am. Chem. Soc.,
113:1026-1030 (1991); and Graycar et al.,
Ann. N.Y. Acad. Sci.,
67:71-79 (1992)). However, while the ratios of esterase-to-amidase activities were improved by these approaches, the absolute esterase activities were lowered concomitantly. Abrahamsen et al.,
Biochemistry,
30:4151-4159 (1991). Chemical modification techniques (Neet et al.,
Proc. Nat. Acad. Sci.,
56:1606 (1966); Polgar et al.,
J. Am. Chem. Soc.,
88:3153-3154 (1966); Wu et al.,
J. Am. Chem. Soc.,
111:4514-4515 (1989); and West et al.,
J. Am. Chem. Soc.,
112:5313-5320 (1990)), which permit the incorporation of unnatural amino acid moieties, have also been applied to improve esterase to amidase selectivity of subtilisins. For example, chemical conversion of the catalytic triad serine (Ser221) of subtilisin to cysteine (Neet et al.,
Proc. Nat. Acad. Sci.,
56:1606 (1966); Polgar et al.,
J. Am. Chem. Soc.,
88:3153-3154 (1966); and Nakatsuka et al.,
J. Am. Chem. Soc.,
109:3808-3810 (1987)) or to selenocysteine (Wu et al.,
J. Am. Chem. Soc.,
111:4514-4515 (1989)), and methylation of the catalytic triad histidine (His 57) of chymotrypsin (West et al.,
J. Am. Chem. Soc.,
112:5313-5320 (1990)) effected substantial improvement in esterase-to-amidase selectivities. Unfortunately however, these modifications were again accompanied by 50- to 1000-fold decreases in absolute esterase activity.
Further, previous attempts to identify enzymes with improved esterase to amidase selectivites have resulted in a slow process which requires large quantities of chemically modified mutant enzymes. In particular, the kinetic constants of fully characterized chemically modified mutant enzymes have been evaluated by the method of initial rates with a colorimetric assay. Amidase activity was followed by the release of p-nitroanilide from the tetrapeptide substrate succinylalanylalanylprolyphenylalanyl p-nitroanilide (sucAAPF-pNa). The analogous thiobenzyl ester substrate succinyl-alanine-alanine-proline-phenylalanine-thiobenzyl ester (sucAAPF-SBn) does not have a chromogenic leaving group, so detection of hydrolysis requires reaction of the thiobenzyl leaving group with 5,5′-dithiobis-2,2′-nitrobenzoate (DTNB, Ellman's reagent). However, the full characterization and kinetic evaluation of new chemically modified mutant enzymes by this method is very material and time-consuming.
The present invention is directed to overcoming these deficiencies.
SUMMARY OF THE INVENTION
The present invention relates to a method for screening chemically modified mutant enzymes for amidase and/or esterase activity. This method includes providing a chemically modified mutant enzyme with one or more amino acid residues from an enzyme being replaced by cysteine residues, where at least some of the cysteine residues are modified by replacing thiol hydrogen in the cysteine residues with a thiol side chain, contacting the chemically modified mutant enzyme with a substrate for an amidase and/or a substrate for an esterase, and determining whether the chemically modified mutant enzyme exhibits amidase and/or esterase activity.
Another aspect of the present invention relates to a chemically modified mutant enzyme with one or more amino acid residues from an enzyme being replaced by cysteine residues, where at least some of the cysteine residues are modified by replacing thiol hydrogen in the cysteine residue with a thiol side chain. The thiol side chain can be —SCH
2
(p-CH
3
—C
6
H
4
), —SCH
2
(p-OCH
3
—C
3
C
6
H
4
), —SCH
2
(p-CF
3
—C
6
H
4
), or —SCH
2
(2,4-diNO
2
—C
6
H
3
).
The present invention also relates to a method of producing a chemically modified mutant enzyme. This method includes providing an enzyme wherein one or more

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