ENZYMES – structure and general characteristics
Enzymes are carriers of many metabolic reactions in the body. Metabolic reactions in the human body would not be possible without enzymes’ presence. Substrates are compounds that are chemically modified by the action of enzymes. The first enzyme isolated in the crystalline state was urease (Summer).
Enzymes contain an active center that is part of their chain and is specially folded. By denaturing the enzyme, they lose their function, although the amino acid sequence remains the same. Many enzymes consist of a protein part and a non-protein (prosthetic) part. Apoenzymes bind such a group reversibly, and the prosthetic group is called a coenzyme.
Enzymes are stereospecific. They react with only one stereoisomer of the substrate.
The properties of enzymes can be divided into 4 rules:
- They reversibly bind the substrate.
- They do not change the direction of the chemical reaction but accelerate it in both directions.
- Chemically unchanged (physically altered) come out of the reaction.
- Enzymatic catalysis can be regulated.
Steps in reactions with enzymes:
The coupling of enzyme and substrate to enzyme-substrate (ES) is an exergonic reaction.
Peter’s law of action applies to every step. Reactions in the body are mostly androgens, and in the presence of enzymes, they are accelerated by a factor of 〖10〗 ^ 8- 〖10〗 ^ 10.
The intermediate reactions by which the overall process occurs are not in balance because one product is always consumed. The second reaction must be exergonic for G = 0 to be valid in total.
Enzymes are mainly globular proteins, molecular Peter from 1000 – 10000. They can combine into multi-enzyme complexes, which become important sites of regulation. The multiple folded part of the chain creates a skeleton to stabilize the active center. At this point, the substrate binds with the release of energy.
The center is located in a groove where side chains of amino acids and sometimes covalently bound substrates can react with the substrate. This structure is not rigid but dynamic and achieves reversible conformational changes. In the binding reaction, the participants’ spatial arrangement is also important, and an induced conformational change of the substrate and enzymes occurs.
Enzymes are mostly isolated in crystalline form, and their structure is determined by X-ray diffraction.
The active site generates multiple portions of the polypeptide chain. The substrate enters the groove, exposed to reactive groups of polypeptide side chains. It can be bound by ionic bond, metal (magnesium ion), hydrophobic, or hydrogen. There is also a conformational change on the substrate and enzyme called induced adaptation.
During the binding reaction, the substrate is removed from the aqueous environment and enters a new one, where a different equilibrium constant, the so-called internal equilibrium constant. The substrate is acted upon by various functional groups in the groove, acting as acceptors or proton donors. Steric and electronic deformation of the substrate occurs.
These above-mentioned processes create a transitional state where the product is also released in addition to the release of energy.
The Ping-Pong mechanism is based on the fact that multiple substrates bind in a certain order. In this process, functional groups transition from one substrate to another. The second substrate binds to the active site only after dissociation of the first. An example of this mechanism is aminotransferases for an amino group transfer, whereby a Schiff base is formed.
In the activation center, some side chains are destined to interact:
Histidine contains an imidazolyl residue, which can receive electrons and pass into the imidazolium ion, forming a ring with two equivalent nitrogen atoms (pKa = 6 – 7). Histidine can be a proton acceptor or donor in a neutral environment, and it is an effective catalyst in acid-base reactions.
Carboxylate ion glutamine and aspartic acid can be proton acceptors.
Arginine has a strongly basic guanidino group that can bind negatively charged groups and thus participate in substrate binding.
Lysine can contribute to substrate binding, and the reactive –NH2 group can form Schiff bases with carbonyl compounds.
Cysteine contains -SH group, which is weakly acidic and is an effective nucleophile. This group reacts with iodoacetamide and N-ethylmaleinimide, and the -SH group is blocked, i.e., enzyme poisoning occurs. Heavy metal ions (Hg, Cu…) and mercury organic compounds also lead to enzyme poisoning. The SH group plays an important role in forming covalent intermediates, such as, e.g., thioesters, which are energy-rich compounds.
Serine contains an –OH group that can participate in covalent catalysis. In a serine protease, the hydroxyl group binds to the rest of the peptide chain. This group can also form hydrogen bonds and thus participate in binding the substrate.
Tyrosine contains a phenolic group which may be a nucleophile in dissociated form, while in the protonated state, it is a participant in hydrophobic interactions with aromatic substrates.
The affinity labeling method synthesizes a substrate that binds to the active site. By cleaving the enzyme with a protease and examining the resulting peptides, it can be determined which amino acids from the active site are involved in substrate binding and other reactions.
Groups that can be reversibly protonated are also found on enzyme proteins’ surfaces. Their degree of dissociation depends on the pH, and therefore the conformation of the protein and its catalytic action change depending on the pH.
The amount of pH at which the catalytic action is best is called pH-optimum.
As the temperature increases, so does the enzyme activity until denaturation begins.
In enzymatic catalysis, due to the catalytic center’s specificity, only one reaction is always catalyzed. Due to the size, conformation, and distribution of the charge, there is a specificity towards the substrate, which can be expressed differently. Specificity to stereoisomers is particularly pronounced and applies to both chiral and non-chiral substrates.
Sometimes several substances react with a single enzyme, so we are talking about group specificity (glycosidases).
Isoenzymes catalyze the same reaction but differ in structure, which can be genetically determined. There are either really different genes or oligomers composed of different subunits. An isoenzyme is aldolase or lactate dehydrogenase, which has 5 different tetramers, and the expression of individual subunits depends on the organ. They may differ from each other in terms of specificity for a substrate or sensitivity to regulatory factors.
Multienzyme complexes are aggregates of several enzymes that catalyze a series of related reactions (pyruvate or oligomeric supra structures are composed in part of enzymatic proteins formed by gene fusion. Their principle of action is that the intermediate of the enzymatic reaction is transferred from one enzyme to another within the complex (supra structure).