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Catalytic Antibodies  
  
2161   11:28 صباحاً   date: 19-12-2015
Author : S. J. Benkovic
Book or Source : Annu. Rev. Biochemistry 61, 29–54.
Page and Part :

Catalytic Antibodies

 

 The concept of catalytic antibodies owes its origin to Pauling and Jencks, who proposed that an antibody normally differs from an enzyme by its inability to bind selectively and to stabilize the transition state of a chemical reaction. An antibody that did happen to be specific for a transition state should therefore function like an enzyme and promote chemical catalysis of the corresponding reaction (1). Advances in chemical synthesis and in hybridoma technology to produce monoclonal antibodies have enabled the exploitation of the diversity and affinity of the immune repertoire to develop catalytic antibodies that are elicited against transition state analogues. The binding sites of these antibodies are anticipated to bind substrates structurally related to the transition state and to process them to products through a pathway lower in free energy, and therefore more rapid, than the normal one that occurs in the absence of antibody. By clever design of appropriate transition state analogues, “tailor-made catalysts” should be created to catalyze reactions with no enzymic counterparts. Challenge of the immune system with a transition state analogue to induce a catalytic antibody is a necessary, but not a sufficient, condition to generate an effective catalyst; factors other than high affinity for the transition state, such as precise orientation of catalytic residues and the effective release of reaction products at the active site, also contribute to the overall catalytic efficiency of an antibody. Strategies have evolved to design transition state analogue immunogens that would solicit catalytic functions within the antibody combining site for efficient catalysis.

1. Reactions Catalyzed by Antibodies

 There are now approximately 100 reactions that have been catalyzed by antibodies (1-3). These reactions include (1) pericyclic processes (oxy-Cope rearrangement, Diels–Alder condensation, Claisen rearrangement), (2) elimination reactions (decarboxylation, dehydration, syn elimination of HF from fluoroketones, E2 (biomolecular) elimination of benisoxazole), (3) hydrolyses (carbonate esters, esters, amide, lactones, enol ethers), (4) bond-forming reactions (lactonization, peptide synthesis, cationic cyclization, aldol condensation), and (5) redox reactions (ketone reduction, epoxidation, sulfoxide oxidation). Antibodies can catalyze, with high stereo- and regiospecificity, reactions that may not generally be catalyzed by enzymes. This property has appeal in the potential application of antibody catalysis to commercial synthetic and medical uses, such as pharmaceutical synthesis and prodrug activation.

 2. The Nature of the Catalytic Site

The active sites of catalytic antibodies elicited by a given immunogen exhibit high sequence homologies and are often structurally convergent (4). These sites harbor shallow clefts complementing the structural and electronic features of the immunogen. The positioning of active site residues in the binding pocket is accomplished by somatic mutation of the germline ancestral antibody arising from an immunological response (5). The germline antibody undergoes a substantial amount of induced-fit conformational change on binding the immunogen. By the process of affinity maturation, the active site residues in the mature antibody become preoriented such that a rigid binding pocket is generated for optimal binding (lock-and-key fit).

 3. Nature of Catalysis

Catalysis by antibodies is like that by enzymes, in that it exhibits Michaelis–Menten kinetics in which substrate binding precedes a chemical transformation, followed by dissociation of the product. Kinetic characterization experiments (6) indicate that the antibody-catalyzed reaction recapitulates a number of characteristics expected of an enzyme, but the chemical transformation step is often rate-limiting. The rate enhancement is generally less than that observed for enzymes catalyzing similar reactions. Transition state analogues are only approximations of the true transition state, and they also do not demonstrate the extremely tight binding to enzymes that is predicted theoretically. Furthermore, antibodies catalyze reactions primarily through restrictions on the translational and rotational movement of substrates, and only to a lesser extent through the active-site acid/base or nucleophilic catalysis that occurs in enzymes. The difference in catalytic efficiency between antibody and enzyme can also be attributed in part to the latter's ability to provide more extensive electrostatic interactions and hydrogen bonding during catalysis through conformational mobility (7).

4. Generation of Catalytic Antibodies

The recovery of catalytic antibodies relies on an efficient means of sampling the immune repertoire and screening for effective catalysts. The murine immune repertoire is estimated to have a diversity of 108, and it can be further expanded by immunization. The majority of catalytic antibodies have been derived from hybridomas that generally capture only 0.01% of the immune repertoire. The development of recombinant antibody fragments as combinatorial libraries consisting of ≥106 members in lambda and in M13 phage has increased the access to the immune repertoire and possibly the recovery of catalytic antibodies. The diversity of an antibody combinatorial library, and potentially the yield of efficient catalytic antibodies, can be further expanded by polypeptide chain-shuffling experiments in which the heavy-chain fragment of a catalytic antibody is allowed to cross with a library of light-chain fragments elicited by the same immunoglobulin, and vice versa (8).

 5.Screening for Catalytic Antibodies

Catalytic antibodies have been identified by their high affinities for the appropriate haptens, as well as their catalytic activities for the desirable reactions. Given the often low activity of catalytic antibodies, sensitive but specific methods have been developed for identifying the catalyst. CatELISA is based on screening for the presence of antibody-catalyzed reaction products using product-specific antibodies as primary antibody indicators in enzyme-linked immunosorbent assay ) ELISA) (9). An alternative method depends on the catalytic release of a reactive species that can be trapped via covalent modification of the phage-displayed antibody, which then permits the recovery of DNA encoding catalytic antibody clones from the phage particles (10). Another method involves the recovery of antibody clones that sustain growth of microorganisms, such as yeast or bacterial auxotrophs, through catalysis of essential biological pathways (11). The host organisms, however, may also impose an additional selection on what catalytic antibodies can be recovered from the screen, since the host will not survive expression of antibodies that are toxic.

References

1. R. A. Lerner, S. J. Benkovic, and P. G. Schultz (1991) Science 252, 659–667

2. S. J. Benkovic (1992) Annu. Rev. Biochemistry 61, 29–54

3. J. R. Jacobsen and P. G. Schultz (1995) Curr. Opin. Struct. Biol. 5, 818–824

4. J. B. Charbonnier et al. (1997) Science 275, 1140–1142

5. G. J. Wedemayer et al. (1997) Science 276, 1665–1669

6. J. D. Stewart et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7404–7409

7. M. R. Haynes et al. (1994) Science 263, 646–652

8. B. Posner et al. (1994) Trends Biochem. Sci. 19, 145–150

9. D. S. Tawfik et al. (1993) Proc. Natl. Acad. Sci. USA 90, 373–377. 

10. K. D. Janda et al. (1997) Science 275, 945–948

11. Y. Tang, J. B. Hicks, and D. Hilvert (1991) Proc. Natl. Acad. Sci. USA 88, 8784–8786.

12. J. A. Smiley and S. J. Benkovic (1994) Proc. Natl. Acad. Sci. USA 91, 8319–8323. 




علم الأحياء المجهرية هو العلم الذي يختص بدراسة الأحياء الدقيقة من حيث الحجم والتي لا يمكن مشاهدتها بالعين المجرَّدة. اذ يتعامل مع الأشكال المجهرية من حيث طرق تكاثرها، ووظائف أجزائها ومكوناتها المختلفة، دورها في الطبيعة، والعلاقة المفيدة أو الضارة مع الكائنات الحية - ومنها الإنسان بشكل خاص - كما يدرس استعمالات هذه الكائنات في الصناعة والعلم. وتنقسم هذه الكائنات الدقيقة إلى: بكتيريا وفيروسات وفطريات وطفيليات.



يقوم علم الأحياء الجزيئي بدراسة الأحياء على المستوى الجزيئي، لذلك فهو يتداخل مع كلا من علم الأحياء والكيمياء وبشكل خاص مع علم الكيمياء الحيوية وعلم الوراثة في عدة مناطق وتخصصات. يهتم علم الاحياء الجزيئي بدراسة مختلف العلاقات المتبادلة بين كافة الأنظمة الخلوية وبخاصة العلاقات بين الدنا (DNA) والرنا (RNA) وعملية تصنيع البروتينات إضافة إلى آليات تنظيم هذه العملية وكافة العمليات الحيوية.



علم الوراثة هو أحد فروع علوم الحياة الحديثة الذي يبحث في أسباب التشابه والاختلاف في صفات الأجيال المتعاقبة من الأفراد التي ترتبط فيما بينها بصلة عضوية معينة كما يبحث فيما يؤدي اليه تلك الأسباب من نتائج مع إعطاء تفسير للمسببات ونتائجها. وعلى هذا الأساس فإن دراسة هذا العلم تتطلب الماماً واسعاً وقاعدة راسخة عميقة في شتى مجالات علوم الحياة كعلم الخلية وعلم الهيأة وعلم الأجنة وعلم البيئة والتصنيف والزراعة والطب وعلم البكتريا.