Chymotrypsin forms

Three essential amino acid residues in the active site of chymotrypsin form a catalytic triad. Which of the following are roles for these residues in catalysis ... 

At Stanford, Harden M. McConnell developed a new technique, called spin labelling, based upon EPR spectroscopy. While carbon-centered free radicals are extremely reactive and short-lived, radical oxides of nitrogen, such as NO and NO2, are moderately stable. McConnell noted that nitroxyl radicals (RR N-O) are extremely stable if R and R are tertiary and can be chemically attached to biological molecules of interest. In 1965, he published the concept of spin labeling and, in 1966, demonstrated that a spin-labelled substrate added to a-chymotrypsin forms a covalent enzyme-substrate complex. The EPR signal was quite broad suggesting restricted motion consistent with Koshland s induced-fit model. In 1971, McConnell published a smdy in which spin labelling indicated flip-flop motions of lipids in cell membranes. This was the start of dynamic smdies of cell membranes. 

Fig. 5. Protein folding. The unfolded polypeptide chain coUapses and assembles to form simple stmctural motifs such as -sheets and a-hehces by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) stmcture in this way. Larger proteins and multiple protein assembhes aggregate by recognition and docking of multiple domains (eg, -barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further stmctural...

Figure 2.19 Organization of polypeptide chains into domains. Small protein molecules like the epidermal growth factor, EGF, comprise only one domain. Others, like the serine proteinase chymotrypsin, are arranged in two domains that are required to form a functional unit (see Chapter 11). Many of the proteins that are involved in blood coagulation and fibrinolysis, such as urokinase, factor IX, and plasminogen, have long polypeptide chains that comprise different combinations of domains homologous to EGF and serine proteinases and, in addition, calcium-binding domains and Kringle domains.

The polypeptide chain of chymotrypsinogen, the inactive precursor of chymotrypsin, comprises 245 amino acids. During activation of chymotrypsinogen residues 14-15 and 147-148 are excised. The remaining three polypeptide chains are held together by disulfide bridges to form the active chymotrypsin molecule. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Figure 11.10 Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3-4 and 5-6, red) of the two domains. These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket.

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Chymotrypsin shows a strong preference for hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan. Flowever, over time chymotrypsin also hydrolyzes amide bonds involving amino acids other than Phe, Tyr, or Trp. Peptide bonds having leucine-donated carboxyls become particularly susceptible. Thus, the specificity... 

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. 

A beta barrel is a three-dimensional protein fold motif in which beta strands connected by loops form a barrellike structure. For example, this fold motif is found in many proteins of the immunoglobulin family and of the chymotrypsin family of serine proteases. 

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. 

To realize the reason for this result from a simple intuitive point of view it is important to recognize that the ionized form of Aspc is more stable in the protein-active site than in water, due to its stabilization by three hydrogen bonds (Fig. 7.7). This point is clear from the fact that the observed pKa of the acid is around 3 in chymotrypsin, while it is around 4 in solution. As the stability of the negative charge on Aspc increases, the propensity for a proton transfer from Hisc to Aspc decreases. 

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

Figure 9-6. Selective proteolysis and associated conformational changes form the active site of chymotrypsin, which includes the Aspl 02-His57-Ser195 catalytic triad. Successive proteolysis forms prochymotrypsin (pro-CT), Jt-chymotrypsin (jt-CT),and ultimately a-chymotrypsin (a-CT), an active protease whose three peptides remain associated by covalent inter-chain disulfide bonds.

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

It is generally accepted that Na ions can be occluded in E]P forms. Occlusion of SNa ions per EP has been demonstrated in chymotrypsin cleaved enzyme and in the Cr-ADP-EiP[3Na] complex [29]. Three Na ions can also be occluded per EP in a complex stabilized by oligomycin in the absence of Mg or phosphate [97] while a maximum of two Na ions are occluded per a subunit in the ouabain complex. 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

The E2 form is not cleaved by chymotrypsin, but trypsin cleaves at Arg (T1) and subsequently at Lys (T2) and tryptic inactivation of E2K or E2P forms is linear and associated with cleavage at Arg" (Ti) [104,108], Inactivation of K-phosphatase is delayed because cleavage of T1 and T2 in sequence is required for inactivation of K-phosphatase activity [105],... 

Powders for Reconstitution. Drugs that have only limited stability in liquid form are prepared as sterile powders for reconstitution by the pharmacist prior to dispensing to the patient. In ophthalmology, these drugs include a-chymotrypsin, echothiophate iodide... 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

---