Reaction chains, enzymic

Addition of a proton (H+) from an acid to a molecule can cause an electron to be withdrawn from one part of the molecule to the part which binds the proton. A base removes a proton from or within a molecnle which will also canse electron shifts. If these shifts favonr formation of the transition state, the rate of the reaction increases. Enzymes possess, in the active site, side-chain gronps of the amino acids that act as acids or bases that is, they can donate or withdraw electrons from the substrate, resulting in electron shifts that favour formation of the transition state (Fignre 3.2). 

By virtue of the amino acid side-chains, enzymes are able to provide a highly specific binding site for their substrates, anchoring these reagents in an appropriate manner and in suitable proximity so that reaction can occur, as well as providing any necessary acid or base catalyst for the reaction. In some cases, a further reagent, a coenzyme, must also be bound for... 

Metabolite flow along a metabolic pathway is mainly determined by the activities of the enzymes involved (see p. 88). To regulate the pathway, it is suf cient to change the activity of the enzyme that catalyzes the slowest step in the reaction chain. Most metabolic pathways have key enzymes of this type on which the regulatory mechanisms operate. The activity of key enzymes is regulated at three independent levels ... 

Finally, the activity of key enzymes can be regulated by ligands (substrates, products, coenzymes, or other effectors), which as allosteric effectors do not bind at the active center itself, but at another site in the enzyme, thereby modulating enzyme activity (6 see p. 116). Key enzymes are often inhibited by immediate reaction products, by end products of the reaction chain concerned feedback inhibition), or by metabolites from completely different metabolic pathways. The precursors for a reaction chain can stimulate their own utilization through enzyme activation. 

An alkylating agent (ICH2COO ) that acts as a potent irreversible inhibitor of enzymes containing reactive thiol, e-amino, and/or imidazole side-chain groups within their active sites. The carboxymethylation reaction with enzymes is typically a bimolecular process that takes place without rate-saturation behavior ... 

The switch function of the GTPase is based on the specific ability of the different functional states of the GTPase to interact with the proteins that precede and follow in the signal chain. A particular GTPase is characterized by the proteins with which the active and inactive forms interact. A special characteristic of the active GTP form is that it may activate effector enzymes further on in the reaction chain, e.g., adenylyl cyclase, and thus actively transmits the signal. 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

Scheme 1. Schematic representation of the system adopted for glucose and pesticide detection. In the upper part of the scheme is shown the reaction chain for the detection of acetylthiocholine giving a measure of acetylcholinesterase (AChE) activity which can be related to pesticide content. In the lower part of the scheme is shown the classic reaction utilised in the case of an oxidase enzyme (glucose oxidase—GOx) for the detection of glucose. In the first case, the final product is thiocholine and in the second, H202, both are measured at the Prussian blue modified electrode at an applied potential of 0.2 V vs. Ag/AgCl and —0.05 V vs. Ag/AgCl, respectively.

In cases where the natural amino acid side chains of enzymes are insufficient to carry out a desired reaction, the enzyme frequently uses coenzymes. A coenzyme is bound by the enzyme along with the substrate, and the enzyme catalyses the bimolecular reaction between the coenzyme and the substrate (cf. Section 2.6.3). A simple model for a-amino acid synthesis by transamination was developed by substituting /I-cyclodextrin with pyridoxamine. Pyridoxamine is able to carry out the transformation of a-keto acids to a-amino acids even without the presence of the cyclodextrin, but with the cyclodextrin cavity as well, the enzyme model proves to be more selective and transaminates substrates with aryl rings bound strongly by the cyclodextrin much more rapidly than those having little affinity for the cyclodextrin. Thus (p-le/f-butylphenyl) pyruvic acid and phenylpyruvic acid are transaminated respectively 15 000 and 100 times faster then pyruvic acid itself, to give (p-le/f-butylphenyl) alanine and phenylalanine (Scheme 12.5). 

Niemeyer CM, Adler M, Blohm D. Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. Anal Biochem 1997 246(1) 140-145. 

Oxidation of acyl-CoA derivatives of fatty acids occurs so that fatty acids are sequentially shortened by two carbon units at a time by a process that yields acetyl-CoA as the only product (Fig. 13-5). The acyl chains are cleaved at the bond between C-2 and C-3 of the chain, which is the so-called /3 bond, by a process that induces oxidation of this part of the molecule. Table 13.1 lists the reactions and enzymes for the /3-oxidation of fatty acids shown in Fig. 13-5. 

Proteolytic enzymes, such as the serine proteases, are among the best characterized of all enzymes.They are important in digestive processes because they break down proteins. They each catalyze the same type of reaction, that is. the breaking of peptide bonds by hydrolysis. The crystal structures of several serine proteases have been determined, and the mechanism of hydrolysis is similar for each. The specificity of each enzyme is, however, different and is dictated by the nature of the side chains flanking the scissile peptide bond (the bond that is broken in catalytic mechanism. Chymotrypsin is one of the best characterized of these serine proteases. The preferred substrates of chymotrypsin have bulky aromatic side chains. The crystal structure determination of the active site of chymotrypsin, illustrated in Figure 18.12, has provided much of the information used to elucidate a plausible mechanism of action of the enzyme. In the first step of any catalyzed reaction, the enzyme and substrate form a complex, ES, the Michaelis complex. The hydrolysis of the peptide bond by chymotrypsin involves three amino acid residues,... 

The copper metalloenzymes are involved in oxygen-using reactions. These enzymes include cytochrome c oxidase (respiratory chain), lysyl oxidase (collagen synthesis), and dopamine [3-hydroxylase (neurotransmitter synthesis). Lysyl oxidase is a small protein with a molecular weight of 32 kDa. This enzyme contains an unusual modification, namely cross-linking between two different parts of its polypeptide chain. The cross-linked region consists of a structure called lysine tyrosylquinone (Klinman, 1996). Two amino acids are involved in this cross-linked structure, and these are Lys 314 and Tyr 349. Lysine tyrosylquinone is used as a cofactor and is necessary for the catalytic activity of the enzyme. Other copper metalloenzymes contain a related cofactor, namely 2,4,5-tiihydrox5q5henylalanine (topaquinone, TPQ). Serum amino oxidase is a copper metalloenzyme that contains TPQ. TPQ consists of a modified residue of phenylalanine. The copper in the active site of the enzyme occurs immediately adjacent to the TPQ cofactor. 

Figure 25.3 Structure of carbamoyl phosphate synthetase. Notice that the enzyme contains sites for three reactions. This enzyme consists of two chains. The smaller chain (yellow] contains a site for glutamine hydrolysis to generate ammonia. The larger chain includes two ATP grasp domains (blue and red). In one ATP-grasp domain (blue), bicarbonate is phosphorylated to carboxyphosphate. which then reacts with ammonia to generate carbarrric acid. In ihe other ATP-grasp domain, the carbamic acid is phosphorylated to produce carbamoyl phosphate. [Drawn from lJDB.pdb.]...

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