Lecture 14 Summary

One of the unifying approaches in biochemistry is to apply a few mechanistic principles to the great variety of enzymes and the reactions they catalyze. This is also where previous attention to the mechanisms in organic chemistry pays real dividends, since (as one respected enzymologist put it) enzymes are not different, just better. Two very common themes in the study of enzymatic reaction mechanisms are general acid/general base catalysis, and nucleophilic attack on electrophilic functional groups. The reactivity of carbonyl compounds and carboxylic acid derivatives underlies the logic of many metabolic reactions. With this précis, the catalytic strategies employed by enzymes, as enumerated and described in our text, are introduced. We follow up with case studies of enzymes with particular emphasis on how their mechanisms fit within our framework and also the modes by which substrate specificity is achieved.

Proteases

Proteases are hydrolase enzymes that catalyze the hydrolysis of a peptide bond in a polypeptide or protein substrate. The peptide bond that is cleaved by a protease is called the scissile bond. The result of a protease-catalyzed hydrolysis of a peptide bond are two separate polypeptides, with new carboxyl and amino termini arising from bond scission.

The four classes of proteases

1. Serine proteases (examples: chymotrypsin, trypsin, elastase; also subtilisin)
2. Cysteine (or thiol) proteases (examples: papain, mammalian cathepsins)
3. Aspartyl proteases (a.k.a. carboxyl or acidic proteases) (examples: pepsin, renin, HIV protease)
4. Metalloproteases (thermolysin, carboxypeptidase A, matrix metalloprotease [MMP])

Chymotrypsin: Covalent catalysis and the double displacement mechanism

The serine and cysteine proteases use a similar mechanism, and we'll treat chymotrypsin [EC 3.4.21.1] as a typical member of the serine proteases. Chymotrypsin follows a double-displacement (or "ping-pong") pattern, which is often observed for enzymes that make use of a covalent catalysis strategy.

The catalytic triad and its role in the serine protease mechanism

The catalytic triad provides a paradigm for the structural and chemical features of enzymes that allow them to facilitate a difficult reaction, peptide bond hydrolysis. The class of enzymes known as serine proteases - of which chymotrypsin is a member - have over the years been very thoroughly characterized by a wide variety of biochemical and structural methods. Residues of chymotrypsin important to its catalytic function were first identified using techniques of protein chemistry such as affinity labels in combination with a suitable assay for the enzyme's activity. Subsequently, the structures of chymotrypsin and other serine proteases revealed that the active sites of these enzymes shared a particular stereochemical arrangement of residues crucial to their activity.

Biochemical investigations of serine proteases

Studies of chymotrypsin using a chromogenic ester substrate show a biphasic kinetics: (1) rapid burst-phase formation of acyl-enzyme intermediate; (2) steady-state phase due to rate-limiting hydrolysis of the acylated enzyme. Note how the kinetics is consistent with a ping-pong mechanism where a step(s) following release of the first product is rate limiting.

Isolation of the acyl-enzyme intermediate: intermediate stable at low pH

Identification of Ser195 - specific labeling of its side chain by reaction with diisopropylphosphofluoridate (DIPF). This serine is unusually reactive, since of 28 serine residues in chymotrypsin, Ser195 is the only serine that is modified by DIPF.

His57 is also catalytically important: affinity labeling by TPCK (tosyl-L-phenylalanine chloromethyl ketone). His57 is alkylated on its ε2 nitrogen of the imidazole ring.

Specificity in serine proteases

The preference of chymotrypsin for large hydrophobic residues at the P1 position of the substrate is accounted for by the existence of an appropriately located, properly sized non-polar cavity in the enzyme, serving as a specificity pocket, well-suited to accommodate the planar aromatic rings of Phe, Tyr, and Trp). In trypsin, this pocket has a negatively charged Asp residue at its bottom, explaining its specificity for Lys and Arg at P1. Elastase shows less well-defined specificity. It tends to prefer smaller, uncharged residues at P1. The elastase specificity pocket is constricted by Val and Thr residues, which replace Gly residues found in trypsin and chymotrypsin.

Convergent evolution in serine proteases

Comparison of the structures of different members of the class of serine proteases reveals close structural and functional similarities. The members within the trypsin family (including chymotrypsin, trypsin, and elastase) show sequence identities as high as 40%, and not surprisingly, are correspondingly similar in structure and functional properties. In a superposition of the main chain traces of chymotrypsin (pdb file 2cha.pdb) and elastase (pdb file 1est.pdb), the differences in positions of corresponding atoms in the best superposition of two structures is computed. For the c-alpha atoms of chymotrypsin and elastase, the root-mean-square difference (RMSD) value is 1.7 Å.

Interestingly, the catalytic triad seen in serine proteases has apparently arisen independently more than once in the course of evolution. The enzyme subtilisin, from the bacterium Bacillus subtilis, has a very similar Ser-His-Asp catalytic triad, despite being quite different in its overall structure. The residues that form the catalytic triad in subtilisin - Asp32, His64, and Ser221 - occur in a different order in the sequence, further evidence of its independent origin. The enzyme serine carboxypeptidase II from wheat germ has the same catalytic triad, and its structure is distinct from either trypsin or subtilisin. The triad in serine carboxypeptidase is composed of Ser146, Asp338, and His397.

Catalytic antibodies

A striking illustration of the principle that enzymes selectively stabilize transition state for the reactions they catalyze, catalytic antibodies were first envisioned by William Jencks in 1969. It was some years before Jencks' idea - that antibodies elicited to recognize haptens resembling the transition state of a reaction would be capable of catalyzing that reaction - could be empirically tested. Once hybridoma technology made monoclonal antibody production routine, many catalytic antibodies were generated, representing an impressively broad variety of reaction types, including some that were not known to exist in nature. Although catalytic antibodies generally fall short in comparison to enzymes in terms of catalytic proficiency (ability to stabilize the transition state) and rate enhancement (k_cat/k_uncat), it is important to bear in mind that catalytic antibodies are generated in a matter of weeks using a fairly simple (at least conceptually!) biotechnological process. Further work on catalytic antibodies can help enzymologists better understand enzyme mechanisms and lead to practical applications in medicine.

Learning objectives

Page update in progress, 10-11-09

References

1. Knowles, JR. (1991) Enzyme catalysis: not different, just better. Nature 350: 121-124.
2. Silverman RB. The Organic Chemistry of Enzyme-Catalyzed Reactions (Revised ed., 2002, Academic Press).
3. Walsh, C. Enzymatic Reaction Mechanisms (1979, WH Freeman and Co.)
4. Jencks WP. Catalysis in Chemistry and Enzymology (1987, Dover)
5. Hilvert D. (2000) Critical Analysis of Antibody Catalysis. Annu Rev Biochem 69: 751-793.
6. Antibodies Molecule of the Month @ Protein Data Bank (this MOM feature includes an example of a catalytic antibody).