Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1997-12-18
2002-10-15
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S221000, C435S222000, C435S252310, C435S320100, C435S471000, C536S023200, C510S300000
Reexamination Certificate
active
06465235
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and to DNA sequences encoding the same. Such mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in a precursor amino acid sequence.
BACKGROUND OF THE INVENTION
Serine proteases are a subgroup of carbonyl hydrolase. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. M. (1974)
Sci Amer.
131, 74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct familites of enzymes: the
Bacillus
subtilisins and the mammalian and homologous bacterial serine proteases (e.g., trypsin and
S. gresius
trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977)
Ann. Rev. Biochem.
46, 331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.
Subtilisin is a serine endoprotease (MW 27,500) which is secreted in large amounts from a wide variety of Bacillus species. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1971) in
The Enzymes
, ed. Boyer P. D., Acad Press, New York, Vol. III, pp. 561-608; Nedkov, P. et al. (1983)
Hoppe-Seyler's Z. Physiol. Chem.
364, 1537-1540. The three-dimensional crystallographic structure of subtilisin BPN′ (from
B. amyloligoefaciens
) to 2.5A resolution has also been reported. Wright, C. S., et al. (1969)
Nature
221, 235-242; Drenth, J. et al. (1972)
Eur. J. Biochem.
26, 177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972)
Biochemistry
11, 2439-2449), product complexes (Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs (Matthews, D. A., et al (1975)
J. Biol. Chem.
250, 7120-7126; Poulos, T. L., et al. (1976)
J. Biol. Chem.
251, 1097-1103), which have been reported have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983)
Mol. Cell. Biochem.
51, 5-32; Svendsen, I. B. (1976)
Carlsberg Res. Comm.
41, 237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methione at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965)
J. Biol. Chem.
244, 5333-5338).
Substrate specificity is a ubiquitous feature of biological macromolecules that is determined by chemical forces including hydrogen bonding, electrostatic, hydrophobic and steric interactions. Jencks, W. P., in
Catalysis in Chemistry and Enzymology
(McGraw-Hill, 1969) pp. 321-436; Fersht, A., in
Enzyme Structure and Mechanism
(Freeman, San Francisco, 1977) pp. 226-287. Substrate specificity studies of enzymes, however, have been limited to the traditional means of probing the relative importance of these binding forces. Although substrate analogs can be synthesized chemically, the production of modified enzyme analogs has been limited to chemically modified enzyme derivatives (Kaiser, E. T., et al. (1985)
Ann. Rev. Biochem.
54, 565-595 or naturally occurring mutants. Kraut, J. (1977)
Ann. Rev. Biochem.
46, 331-358.
The recent development of various in vitro techniques to manipulate the DNA sequences encoding naturally-occuring polypeptides as well as recent developments in the chemical synthesis of relatively short sequences of single and double stranded DNA has resulted in the speculation that such techniques can be used to modify enzymes to improve some functional property in a predictable way. Ulmer, K. M. (1983)
Science
219, 666-671. The only working example disclosed therein, however, is the substitution of a single amino acid within the active site of tyrosyl-tRNA synthetase (Cys35→Ser) which lead to a reduction in enzymatic activity. See Winter, G., et al. (1982)
Nature
299, 756-758; and Wilkinson, A. J., et al. (1983)
Biochemistry
22, 3581-3586 (Cys35→Gly mutation also resulted in decreased activity).
When the same t-RNA synthetase was modified by substituting a different amino acid residue within the active site with two different amino acids, one of the mutants (Thr51→Ala) reportedly demonstrated a predicted moderate increase in kcat/Km whereas a second mutant (Thr51→Pro) demonstrated a massive increase in kcat/Km which could not be explained with certainty. Wilkinson, A. H., et al. (1984)
Nature
307, 187-188.
Another reported example of a single substitution of an amino acid residue is the substitution of cysteine for isoleucine at the third residue of T4 lysozyme. Perry, L. J., et al. (1984)
Science
226, 555-557. The resultant mutant lysozyme was mildly oxidized to form a disulfide bond between the new cysteine residue at position 3 and the native cysteine at position 97. This crosslinked mutant was initially described by the author as being enzymatically identical to, but more thermally stable than, the wild type enzyme. However, in a “Note Added in Proof”, the author indicated that the enhanced stability observed was probably due to a chemical modification of cysteine at residue 54 since the mutant lysozyme with a free thiol at Cys54 has a thermal stability identical to the wild type lysozyme.
Similarly, a modified dehydrofolate reductase from
E. coli
has been reported to be modified by similar methods to introduce a cysteine which could be crosslinked with a naturally-occurring cysteine in the reductase. Villafranca, D. E., et al. (1983)
Science
222, 782-788. The author indicates that this mutant is fully reactive in the reduced state but has significantly diminished activity in the oxidized state. In addition, two other substitutions of specific amino acid residues are reported which resulted in mutants which had diminished or no activity.
As set forth below, several laboratories have also reported the use of site directed mutagensis to produce the mutation of more than one amino acid residue within a polypeptide.
The amino-terminal region of the signal peptide of the prolipoprotein of the
E. coli
outer membrane was stated to be altered by the substitution or deletion of residues 2 and 3 to produce a charge change in that region of the polypeptide. Inoyye, S., et al. (1982)
Proc. Nat. Acad. Sci. USA
79, 3438-3441. The same laboratory also reported the substitution and deletion of amino acid redisues 9 and 14 to determine the effects of such substitution on the hydrophobic region of the same signal sequence. Inouye, S., et al. (1984)
J. Biol. Chem.
259, 3729-3733. In the case of mutants at residues 2 and 3 the authors state that the results obtained were consistant with the proposed loop model for explaining the functions of the signal sequence. However, as reported the mutations at residues 9 and 14 produced results indicating that the signal peptide has unexpeded flexibility in terms of the relationship between its primary structure and function in protein secretion.
Double mutants in the active site of tyrosyl-t-RNA synthetase have also been reported. Carter, P. J., et al. (1984)
Cell
38, 835-840. In this report, the improved affinity of the previously described Thr51→Pro mutant for ATP was probed by producing a second mutation in the active site of the
Bott Richard Ray
Caldwell Robert Mark
Cunningham Brian C.
Estell David Aaron
Power Scott Douglas
Flehr Hohbach Test Albritton & Herbert LLP
Genenco International, Inc.
Moore William W.
Patterson Jr. Charles L.
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