Catalytic Residues and Mechanism

Substrate Access and Product Egress Through y9-propellers 

The chloromethyl ketone-based inhibitor-complex crystal structures suggested the strand E993-P996 as a recognition strand for the unprimed-substrate residues. 

The unprimed side of the substrate is held in place by a series of interactions with the protein. In addition to the observed ionic (D936) or hydrophobic SI interaction site (Y946, 1969, V991, F1013), the PI main chain is held by its interaction with the oxyanion hole (G918, D966). P2-P4 residues will presumably utilize un- 

Similarly, there is evidence for functional but not physical interaction of tricorn with the proteasome (Tamura et al. 1998) A physical interaction between these molecules by aligning their respective central pores would imply a symmetry mismatch. While such a physical interaction would be consistent with the geometric dimensions of both molecules, its existence needs to be experimentally confirmed and characterized. 

---

As indicated in Table 4.12, four regions which constitute the catalytic regions of amylolytic enzymes are conserved in the starch-branching isoenzymes of maize endosperm, rice seed and potato tuber, and the glycogen-branching enzymes of E. coli.286,281 It would be of interest to know whether the seven highly conserved amino acid residues of the a-amylase family listed in bold letters in Table 4.12 are also functional in branching enzyme catalysis. Further experiments, such as chemical modification and analysis of the three-dimensional structure of the BEs, would be needed to determine the nature of its catalytic residues and mechanism. 

Vallmitjana, M., Ferrer-Navarro, M., Planell, R., Abel, M., Ausin, C., Querol, E., Planas, A. and Perez-Pons, J. A. (2001) Mechanism of the family 1 /J-glucosidasc from Streptomyces sp Catalytic residues and kinetic studies, Biochemistry 40, 5975-5982. 

Particular role of several catalytic residues, and one-proton vs. two-proton mechanisms... 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Catalytic site of lipase is known to be a serine-residue and lipase-catalyzed reactions are considered to proceed via an acyl-enzyme intermediate. The mechanism of lipase-catalyzed polymerization of divinyl ester and glycol is proposed as follows (Fig. 3). First, the hydroxy group of the serine residue nucleophilically attacks the acyl-carbon of the divinyl ester monomer to produce an acyl-enzyme intermediate involving elimination of acetaldehyde. The reaction of the intermediate with the glycol produces 1 1 adduct of both... 

Fischer, F., and Fetzner, S., Site-Directed Mutagenesis of Potential Catalytic Residues in lh-3-Hydroxy-4-Oxoquinoline 2, 4-Dioxygenase, and Hypothesis on the Catalytic Mechanism of 2, 4-Dioxygenolytic Ring Cleavage. FEMS Microbiol Lett, 2000. 190 pp. 21-27. 

It follows from the above that MPO may catalyze the formation of chlorinated products in media containing chloride ions. Recently, Hazen et al. [172] have shown that the same enzyme catalyzes lipid peroxidation and protein nitration in media containing physiologically relevant levels of nitrite ions. It was found that the interaction of activated monocytes with LDL in the presence of nitrite ions resulted in the nitration of apolipoprotein B-100 tyrosine residues and the generation of lipid peroxidation products 9-hydroxy-10,12-octadecadienoate and 9-hydroxy-10,12-octadecadienoic acid. In this case there might be two mechanisms of MPO catalytic activity. At low rates of nitric oxide flux, the process was inhibited by catalase and MPO inhibitors but not SOD, suggesting the MPO initiation. 

The partitioning of the system in a QM/MM calculation is simpler if it is possible to avoid separating covalently bonded atoms at the border between the QM and the MM regions. An example is the enzyme chorismate mutase [39] for which the QM region could include only the substrate, because the enzyme does not chemically catalyze this pericyclic reaction. In studies of enzyme mechanisms, however, this situation is exceptional, and usually it will be essential, or desirable, to include parts of the protein (for example catalytic residues) in the QM region of a QM/MM calculation, i.e. the boundary between the QM and MM regions will separate covalently bonded atoms (Fig. 6.1). 

The mechanism by which serine peptidases, particularly serine endopep-tidases (EC 3.4.21), hydrolyze peptide bonds in peptides and proteins has been extensively investigated by X-ray crystallography, site-directed mutagenesis, detection of intermediates, chemical modification, H-NMR spectroscopy, and neutron diffraction [2-14], These studies revealed that all serine peptidases possess a catalytic triad, composed of a serine, a histidine, and an aspartate residue, and a so-called oxyanion hole formed by backbone NH groups. 

Fig. 20. Frame-by-frame series of stop-action pictures of the catalytic mechanism of RNase A at atomic resolution. Only the essential active site residues and the substrate (filled bonds) are shown. Frame 1, l e native enzyme. The sulfate ion which binds to the active site is shown. Frame 2, The Michaelis E-S complex with the dinucleotide CpA. The 2 oxygen which is deprotonated by His-12 is blackened. Frame 3, The transition state for...

Contacts with the catalytic residues, in combination with hydrophobic interactions, are also observed in the complex of an insect a-amylase with the Ragi bifunctional a-amylase/trypsin inhibitor (RBI) [174]. Conversely, the mechanism of inhibition of barley a-amylase by the barley a-amylase/subtilisin inhibitor (BASI) did not involve direct contact between inhibitor residues and the catalytic site [175]. The inhibitor sterically blocks the catalytic site, but does not extend into it. A cavity is created, which is occupied by a calcium ion coordinated by water-mediated interactions with the catalytic residues. 

---