Acid-base catalysis substrate structure

Even with the uncertainty in E2 active-site position, the models have suggested that there would be no E3 residues near the E2 active site, in agreement with the observations made in the c-Cbl-E2 structure. This again ruled out the possibility that the SCF E3 provides acid/base catalysis and the possibility that the SCF positions the -amino group of the lysine at the E2 active site [66]. The only plausible mechanism left accounting for the catalysis mediated by the SCF in substrate ubiq-uitination is that the E3 complex helps increase the effective concentration of a portion of the substrate that contains the physiological ubiquitination-site lysine at the E2 active site. This model made the testable prediction that the distance between the destruction motif and the ubiquitinated lysine is a determinant of the ubiquiti-nation efficiency. 

No large conformational changes occur in the enzyme during catalysis, but many small movements take place. The structural basis for the catalytic power of ribonuclease thus resides in several different features tight, specihc binding of a strained conformation of the substrate, general acid-base catalysis by His-12 and His-119, and preferential stabilization of the transition state by ionic interactions with Lys-41. 

For ribonuclease A the occurrence of conformational changes and the occurrence of acid-base catalysis has been well documented. The overall mechanism can be envisaged as follows. The enzyme exists in dynamic equilibrium between two forms differing in the structure of the active site groove. The substrate is bound almost as rapidly as it can diffuse to the active site. Binding of the substrate induces a conformational change that... 

In addition to participating in acid-base catalysis, some amino acid side chains may enter into covalent bond formation with substrate molecules, a phenomenon that is often referred to as covalent catalysis.174 When basic groups participate this may be called nucleophilic catalysis. Covalent catalysis occurs frequently with enzymes catalyzing nucleophilic displacement reactions and examples will be considered in Chapter 12. They include the formation of an acyl-enzyme intermediate by chymotrypsin (Fig. 12-11). Several of the coenzymes discussed in Chapters 14 and 15 also participate in covalent catalysis. These coenzymes combine with substrates to form reactive intermediate compounds whose structures allow them to be converted rapidly to the final products. 

Acid—base catalysis is caused by the formation of a reactive intermediate from substrate and catalyst which opens a low free energy pathway for the reaction. Consequently, the phenomenon of catalysis cannot be separated from problems of reaction mechanism. In this section, various possibilities of reaction mechanisms involving acid—base catalysis are discussed from a deductive point of view, with respect to structure and reactivity of substrates and intermediates [3, 4,14, 83]. 

Enzymes adopt conformations that are structurally and chemically complementary to the transition states of the reactions that they catalyze. Sets of interacting amino acid residues make up sites with the special structural and chemical properties necessary to stabilize the transition state. Enzymes use five basic strategies to form and stabilize the transition state (1) the use of binding energy, (2) covalent catalysis, (3) general acid-base catalysis, (4) metal ion catalysis, and (5) catalysis by approximation. Of the enzymes examined in this chapter, three groups of enzymes catalyze the addition of water to their substrates but have different requirements for catalytic speed and specificity, and a fourth group of enzymes must prevent reaction with water. 

The information within an enzyme s active site (its shape and charge distribution) constrains the motions and allowed conformations of the substrate, making it appear more like the transition state. In other words, the information in the structure of the enzyme is used to optimally orient the substrate. As a result of this information transfer, the energy of the enzyme-substrate complex becomes closer to the AG, which means that the energy needed for the reaction to proceed to product is reduced. Consequently there is an increase in the rate of the enzyme-catalyzed reaction. Other factors, such as electrostatic effects, general acid-base catalysis, and covalent catalysis (discussed on pp. 177-180), also contribute to the increased rates of enzyme-catalyzed reactions over non-enzyme catalyzed reactions. 

Hydrogen transfer is one of the most pervasive and fundamental processes that occur in biological systems. Examples include the prevalent role of acid-base catalysis in enzyme and ribozyme function, the activation of C-H bonds leading to structural transformations among a myriad of carbon-based metabolites, and the transfer of protons across membrane bilayers to generate gradients capable of driving substrate transport and ATP biosynthesis. 

Figure 13 Structures of PTPs include two important motifs, the P-loop that bears the cysteine nucleophile within the general signature motif (H/V)Cp<)5R(S/T), and the WPD-loop, which includes an important aspartic acid, a general acid-base catalyst. Substrate binding by the P-loop promotes a change of the WPD-loop conformation from an open, inactive to a closed, active conformation in which the aspartic acid completes the catalytic ensemble used for catalysis. The representation in this figure was created using PyMol from the PTP1B structures in apo-bound (PDB 2CM2) and inhibitor-bound (PDB 1BZJ) forms.

The WPD loop is a flexible /3-turn found in all tyrosine-specific PTPs, and includes the conserved aspartic acid residue that serves as a general acid-base catalyst. Substrate binding thermodynamically favors the closed, catalytically active conformation, where the aspartic acid is in position for catalysis (Figure 15). The DSPs also share a conserved aspartic acid in this catalytic role. However, except for VHZ, a recently purified DSP which may possess a flexible IPD loop, the aspartic acid in DSPs is located on a rigid structure. Consequently, no conformational change analogous to WPD loop movement in PTPs seems to be associated with catalysis for most DSPs. 

Usually p and q are taken to refer to different atoms in the same molecule, so that, for example, p is taken as 2 for fumaric acid but as 1 for ammonium ion (even though 4 would seem appropriate). Equations (8-47) and (8-48) are applicable only if structurally and electrostatically similar acid and base catalysts are compared. Note that Eq. (8-38) can be cast into an essentially identical form since the pK of the proton acceptor, the substrate, is constant for acid catalysis, and the pK of the proton donor, the substrate, is constant for base catalysis. This is not too surprising, since a proton-transfer reaction is often rate-determining in acid-base catalysis. 

The impact of nucleophilic and electrophilic groups of the active center on the substrate at the contact area in the enzyme-substrate complex (the effect of synchronous intramolecular catalysis). The polyfunctional catalysis involves a great many processes push-pull mechanisms, processes involving a relay charge transfer, as well as a general acid-base catalysis. Presumably, the enzyme in the initial state of the enzymatic reaction already contains structural elements of the transition state and in this case the reaction must be thermodynamically more advantageous. 

Another promising system involves the concerted acid/base catalysis of enolization of ketones. Stereoelectronic considerations [25] indicate that an optimal catalyst for this reaction requires that acid and base components converge from perpendicular directions on the ketone (Scheme 12). The structure 27 (a glycine derivative of Kemp s triacid) exhibits considerable activity in the enolization of phenylacetone. It should be possible to engineer additional points of contact between ketone (substrate) and diacid (catalyst) to enhance the enolization process, and we are working toward this goal. 

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