Active site enzyme mechanism

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

Mimetic catalysis designs a real model (a mimic) which simulates objects and processes of enzymatic catalysis by their basic (but deficient) characteristics (selectivity, mildness of condition, active site action mechanism, etc.). Since only definite properties of the enzyme are simulated, it does not profess to a complete enzyme description, though optimal parameters by some properties may be approached. The mimetic model of enzyme helps in synthesizing suitable catalysts using inaccurate and sometimes ambiguous information. 

Leucine aminopeptidase is interesting in that its active site contains two zinc atoms which together bind and activate the water molecule [74]. Despite this enzyme containing a dinuclear metal center at its active site, its mechanism, and specifically its mode of proton transfers reactions, appear to follow the general theme established by thermolysin and carboxypeptidase Adenosine deaminase and other members of the family of nucleoside and nucleotide deaminases utilize zinc-bound water as the catalytic nucleophile to displace ammonia from the 6-position of purines or the 4-position of pyrimidines and in all cases display inverse solvent deuterium isotope effects ranging from 0.3 to 0.8 on fec/Kni [75-80]. These effects are reminiscent of those observed for metallopro-teases and have their origins, like those of the proteases, in fractionation factors for the protons of the bound water that are less than one. 

To illustrate how the active site binds a specific substrate and then promotes a chemical change in the bound substrate, we examine the action of cyclic AMP-dependent protein kinase, now generally referred to as protein kinase A (PKA). This enzyme and other protein kinases, which add a phosphate group to serine, threonine, or tyrosine residues in proteins, are critical for regulating the activity of many cellular proteins, often in response to external signals. Because the eukaryotic protein kinases belong to a common superfam-lly, the structure of the active site and mechanism of phosphorylation are very similar in all of them. Thus protein kinase A can serve as a general model for this important class of enzymes. 

The PLP resides as an internal aldimine forming a Schiff base with a Lys residue in the active site. The mechanism is predicted to involve the formation of PLP-bound species, in analogy with other enzymes... 

Storage proteins are hydrolysed into their constituent amino acids by proteinases (proteases), enzymes which have been classified into four major groups based on their active site catalytic mechanisms. These are ... 

These enzymes have in common the presence of a serine and a histidine residue in their active sites (for mechanism, see 2.4.2.5). 

The estimated value of the worldwide sales of industrial enzymes was 1 billion in 1998 (Anwar and Saleemuddin, 1998), 1.5 billion in 2000 and 3.3 billion in 2010 (Sarrouh et al., 2012). Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total worldwide sale (Figure 9.1). They differ in the properties such as substrate specificity, active sites, catalytic mechanism, pH, temperature and activity profiles. Figure 9.2 presents a breakdown of the market shares for industrial proteases. These data clearly show that bacterial proteases are the significant segment representing 60% of the total industrial protease turnover. 

High-resolution n.m.r. combined with information from X-ray diffraction, is revealing much about the active sites and mechanisms of action of various enzymes, such as chymotrypsin (Gerig, 1968) and lysozyme (Cohen and Jardetzky, 1968), and the same benefits are expected for drugs and their receptors. Selective deuteration of the enzyme is a great help in these studies (Putter et al.y 1969). 

For many applications, especially studies on enzyme reaction mechanisms, we do not need to treat the entire system quantum mechanically. It is often sufficient to treat the center of interest (e.g., the active site and the reacting molecules) quantum mechanically. The rest of the molecule can be treated using classical molecular mechanics (MM see Section 7.2). The quantum mechanical technique can be ab-initio, DFT or semi-empirical. Many such techniques have been proposed and have been reviewed and classified by Thiel and co-workers [50] Two effects of the MM environment must be incorporated into the quantum mechanical system. 

Molecular volumes are usually computed by a nonquantum mechanical method, which integrates the area inside a van der Waals or Connolly surface of some sort. Alternatively, molecular volume can be determined by choosing an isosurface of the electron density and determining the volume inside of that surface. Thus, one could find the isosurface that contains a certain percentage of the electron density. These properties are important due to their relationship to certain applications, such as determining whether a molecule will fit in the active site of an enzyme, predicting liquid densities, and determining the cavity size for solvation calculations. 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Elucidating Mechanisms for the Inhibition of Enzyme Catalysis An inhibitor interacts with an enzyme in a manner that decreases the enzyme s catalytic efficiency. Examples of inhibitors include some drugs and poisons. Irreversible inhibitors covalently bind to the enzyme s active site, producing a permanent loss in catalytic efficiency even when the inhibitor s concentration is decreased. Reversible inhibitors form noncovalent complexes with the enzyme, thereby causing a temporary de-... 

---