Catalytic hydrolases

Some G proteins are slow GTP hydrolases with turnover numbers around two per minute, others such as Ras are only marginally catalytic. Kinetic experiments in solution have shown that in both cases the most likely mechanism... 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

An enzyme in which the single catalytic residue is at the N-terminus of the protein. Many Ntn-hydrolases are synthesized as precursors and autoactivate the precursors are therefore peptidases, even if the mature enzyme has no further proteolytic activity. Three of the beta subunits of the proteasome are Ntn-hydrolases. 

SxxK (3-lactamases are uncoupled SxxK acyl transferases that work as (3-lactam antibiotic hydrolases. They represent a mechanism of defence of great efficiency. On good (3-lactam substrates, their catalytic centres can turn over 1000 times per second. 

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

Figure 2.14 CASTing of the epoxide hydrolase from A. niger (ANEH) based on the X-ray structure of the WT [61]. (a) Defined randomization sites A-E (b) top view of tunnel-like binding pocket showing sites A-E (blue) and the catalytically active D192 (red) [23].

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

In this article are discussed the results of those studies which have become available over the past 15 years and which permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities. It will be seen that, with few exceptions, the data support a mechanism almost identical to that proposed by Phillips and his group for lysozyme. ... 

Gladyshev VN, SV Khangulov, TC Stadtmann (1994) Nicotinic acid hydrolase from Clostridium barkeri electron paramagnetic studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc Natl Acad USA 91 232-236. 

Rink R, M Eennema, M Smids, U Dehmel, DB Janssen (1997) Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter ADI. J Biol Chem 272 14650-14657. 

Adenosine deaminase (ADA) is an amino hydrolase that catalyzes the deamination of adenosine and 2 -deoxyadenosine to inosine and 2 -deoxyinosine, respectively. High activity of ADA is seen in thymus and other lymphoid tissues. ADA has been shown in many different physical forms. A small form of the enzyme predominates in the spleen, stomach, and red blood cells, whereas the large form predominates in the kidney, liver, and skin fibroblasts. The small form of the catalytic subunit can be converted to the large form by complexing with a protein termed binding protein or complexing protein. 

The matrix metalloprotease (MMP) family of zinc hydrolases are thought to play important roles in extracellular tissue remodeling in angiogenesis and other normal physiological processes, in some inflammatory processes and in metastatic processes in cancer. Like the zinc carboxypeptidases, the MMPs also utilize a zinc-coordinated water molecule to initiate attack on the scissile amide bond of protein substrates. These enzymes are synthesized by the ribosome in a latent form composed of a catalytic domain and an N-terminal extension, referred to as the prodomain the latent, or inactive form of the enzyme is referred to as a zymogen or... 

Gong, P.-F., Xu, J.-H, Tang, Y.-F. and Wu, H.-Y. (2003) Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80. Biotechnology Progress, 19, 652-654. 

Remarkably, Brassica napus pollen was reported to have a 22 kDa cutinase that cross-reacted with antibodies prepared against F. solani f. pisi cutinase [134]. Although a 22 kDa and a 42 kDa protein that catalyzed hydrolysis of p-nitrophenyl butyrate were found in this pollen, only the former catalyzed cutin hydrolysis. Immunofluorescence microscopic examination suggested that the 22 kDa protein was located in the intine. Since the nature of the catalytic mechanism of this enzyme has not been elucidated, it is not clear whether this represents a serine hydrolase indicating that plants may have serine and thiol cutinases. The role of the pollen enzyme in controlling compatibility remains to be established. 

The primary and tertiary structures of the cholinesterases are known. The primary structures of the cholinesterases initially defined a large and functionally eclectic superfamily of proteins, the a,P hydrolase fold family, that function not only catalytically as hydrolases but also as surface adhesion molecules forming heterologous cell contacts, as seen in the structurally related proteins... 

The role of the serine residue in hydrolysis was further examined using pseudo-substrates, e.g. p-nitrophenylacetate—substrates which were only very slowly utilized by the enzyme. The p-nitrophenyl group was slowly released and the acyl group became attached to the same serine in hydrolases which had been detected by DIPF (Kilby and Youatt, 1954). Mechanisms for peptide and ester hydrolysis were therefore proposed in which the acyl group became transiently and covalently bound to serines in catalytic sites (see Hartley et al. 1969). 

The previous chapter offered a broad overview of peptidases and esterases in terms of their classification, localization, and some physiological roles. Mention was made of the classification of hydrolases based on a characteristic functionality in their catalytic site, namely serine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallopeptidases. What was left for the present chapter, however, is a detailed presentation of their catalytic site and mechanisms. As such, this chapter serves as a logical link between the preceding overview and the following chapters, whose focus is on metabolic reactions. 

Fig. 3.2. Common catalytic groups of hydrolases involved in ester and amide bond hydrolysis (Z+ = electrophilic component polarizing the carbonyl group Y = nucleophilic group attacking the carbonyl C-atom H-B = proton donor transforming the -OR or -NR R" moiety into...

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