Chemically modified enzymes with multiple charged variants

Details Chemically modified enzymes with multiple charged variants Chemically modified enzymes with multiple charged variants

1999-12-20

2002-04-30

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C510S392000

Reexamination Certificate

active

06379942

ABSTRACT:

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[ Not Applicable ]
FIELD OF THE INVENTION
This invention relates to the field of chemically modified mutant enzymes. In particular this invention pertains to chemically modified mutant enzymes in which multiply charged substituents are introduced to enhance interaction of the enzyme with a charged substrate.
BACKGROUND OF THE INVENTION
For both protein chemistry (Nilsson et al. (1992)
Curr. Opin. Struct. Biol
2: 569-575; LaVallie and McCoy (1995)
Curr. Opin. Biotechnol.
6: 501-506; Uhlen and Moks (1990)
Methods Enzymol.
185: 129-143) and organic synthesis applications (Sears and Wong (1996)
Biotechnol. Prog.
12: 423-433; Faber (1997)
Biotransformations in Organic Synthesis:
3rd ed. Springer-Verlag: Heidelberg; Roberts (1993)
Preparative Biotransformations; Wiley: New York:
1993) it is desirable to have available a diverse toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. To date the most extensively exploited class of enzymes in organic synthesis applications have been the hydrolases. Among these, the serine proteases have received considerable attention due, in part, to their often exquisite stereo-, regio-, and chemo-selectivities (Sears and Wong (1996)
Biotechnol. Prog.
12: 423-433; Faber (1997)
Biotransformations in Organic Synthesis:
3rd ed. Springer-Verlag: Heidelberg; Roberts (1993)
Preparative Biotransformations
; Wiley: New York: 1993; Moree et al. (1997)
J. Am. Chem. Soc.
119: 3942-3947).
While over 3000 enzymes have now been reported, of which many are proteases, significantly fewer of the latter are available inexpensively from commercial sources (Faber (1997)
Biotransformations in Organic Synthesis:
3rd ed. Springer-Verlag: Heidelberg; Roberts (1993)
Preparative Biotransformations
; Wiley: New York: 1993; Moree et al. (1997)
J. Am. Chem. Soc.
119: 3942-3947; Jones (1986)
Tetrahedron
42: 3351-3403). Furthermore, since wild type enzymes do not accept all substrate structures of synthetic interest, it is attractive to contemplate the tailoring of a readily available protease in order to expand their substrate specificities in a controlled manner with the ultimate goal of creating any desired specificity at will.
In this regard, the goal of specificity alteration of enzymes has already been targeted by several different approaches. For example, site-directed mutagenesis (Perona and Craik (1995)
Protein Sci.
4: 337-360) and random mutagenesis (Arnold (1998)
Acc. Chem. Res.
31(3): 125-131) have been employed to tailor enzyme specificity and have permitted some insights into the electrostatic (Wells et al. (1987)
Proc. Natl. Acad. Sci. USA,
84: 5167-5171; Wells et al. (1987)
Proc. Nat. Acad. Sci. USA,
84: 1219-1223; Wells and Estell (1988)
TIBS
13: 291-297; Bott et al. (1987) Pages 139-147 In:
Biotech. Agric.Chem.
; Lebanon, Mumma, Honeycutt, Duesing, eds.; Vol. ACS Symp. Ser. 334; Russell et al. (1987)
J. Mol. Biol.
193: 803-813; Ballinger et al. (196)
Biochemistry
33: 13579-13585), steric (Rheinnecker et al. (1994)
Biochemistry
33: 221-225; Rheinnecker et al. (1993)
Biochemistry
32(5): 1199-1203; Sørensen et al. (1993)
Biochemistry
32: 8994-8999; Estell et al. (1986)
Science
233: 659-663; Takagi et al. (1996)
FEBS Lett.
395: 127-132; Takagi et al. (1997)
Protein Eng.
10(9): 985-989), and hydrophobic (Estell et al. (1986)
Science
233: 659-663; Wangikar et al. (1995)
Biochemistry
34(38): 12302-12310; Bech et al. (1993)
Biochemistry
32: 2845-2852) factors which govern enzyme-substrate interactions. However, the structural variations within these approaches are limited to the 20-natural amino acids. Consequently, biosynthetic methods have recently been developed to introduce unnatural amino acids into proteins (25. Cornish et al. (1995)
Angew. Chem. Int. Ed.Eng.
34: 621-633; Parsons et al. (1998)
Biochemistry
37: 6286-6294; Hohsaka et al. (1996)
J. Am. Chem. Soc.
118(40): 9778-9779). Unnatural functionalities have also been incorporated by chemical modification techniques (Kuang et al. (1996)
J. Am. Chem. Soc.
118: 10702-10706; Ory et al. (1998)
Protein. Eng.
11(4): 253-261; Peterson: E. B.; Hilvert: D.
Biochemistry
34: 6616-6620; Suckling: C. J.; Zhu: L.-M.
Bioorg. Med. Chem. Lett.
3: 531-534; Rokita and Kaiser (1986)
J. Am. Chem. Soc.
108: 4984-4987; Kokubo et al. (1987)
J. Am. Chem. Soc.
109: 606-607; Radziejewski et al. (1985)
J. Am. Chem. Soc.
107: 3352-3354). Generally, however, unnatural amino acid mutagenesis approach is not yet amenable to large scale preparations, and chemical modification alone is insufficiently specific.
SUMMARY OF THE INVENTION
This invention provides novel multiply-charged chemically modified mutant enzymes. In a particularly preferred embodiment this invention provides a modified enzyme where one or more amino acid residues in the enzyme are replaced by cysteine residues. The cysteine residues are modified by replacing the thiol hydrogen in the residue with a substituent group providing a thiol side chain comprising a multiply charged moiety. Preferred enzymes include serine hydrolases, more preferably proteases (e.g. subtilisins). One particularly preferred enzyme is a
Bacillus lentus
subtilisin.
The amino acid replaced with a cysteine may include an amino acid selected from the group consisting of asparagine, leucine, methionine, and serine. Preferred replaced amino acids are in a binding site (e.g., a subsite such as S1, S1′, and S2). Where the enzyme is a subtilisin-type serine hydrolase the cysteine(s) is substituted amino acid(s) corresponding to a
Bacillus lentus
subtilisin residue selected from the group consisting of residue 156, reside 166, residue 217, residue 222, residue 62, residue 96, residue 104, residue 107, reside 189, and residue 209. Where the enzyme is a trypsin-chymotrypsin-type serine protease the cysteine(s) are substituted for and amino acid corresponding to a trypsin residue selected from the group consisting of Tyr94, Leu99, Gln175, Asp189, Ser190, and Gln192. Where the enzyme is an alpha/beta serine hydrolase the cysteine(s) are substituted for and amino acid corresponding to a
Candida antartica
lipase (protein Data Bank entry 1tca) residue selected from the group consisting of Trp104, Thr138, Leu144, Val154, Ile189, Ala225, Leu278 and Ile185.
The multiply charged moiety can be negatively or positive charged and in certain embodiments, the enzyme can contain both positively and negatively multiply charged moieties. Particularly preferred negatively charged moieties include, but are not limited to, sulfonatoethyl thiol, 4-carboxybutyl thiol, 3,5-dicarboxybenzyl thiol, 3,3-dicarboxybutyl thiol, and 3,3,4-tricarboxybutyl thiol, while particularly preferred positively charged moieties include, but are not limited to, amingethyl thiol, 2-(trimethylammonium)ethyl thiol, 4,4-bis(aminomethyl)-3-oxo-hexyl thiol, and 2,2-bis(aminomethyl)-3-aminopropyl thiol. The multiply charged moiety can also be a dendrimer or a polymer.
In another embodiment, this invention provides methods of making novel multiply-charged chemically modified mutant enzymes. The methods involve providing an enzyme having one or more amino acids have been replaced with cysteine residues; and replacing the thiol hydrogen, in one or more cysteine residues, with a substituent group providing a thiol side chain comprising a multiply charged moiety. In certain embodiments, a native cysteine can be chemically modified and there is no need to introduce a cysteine. Preferred enzymes include serine hydrolases as identified herein. Preferred residues for replacement with a cysteine and preferred multiply-charged moieties are identified herein.
In another embodiment, this invention includes a composition comprising any one of the multiply charged chemically modified mutant enzymes as described herein and a detergent or other cleaning agent.
In still another embodiment, this invention provides methods of assaying for a preferred enzyme.

Affiliated with

Also associated with

Rating

Chemically modified enzymes with multiple charged variants does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Chemically modified enzymes with multiple charged variants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chemically modified enzymes with multiple charged variants will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2898891