Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Enzymes
A Brief History of the Development of Enzymology
Chemical Nature of Enzymes

Today, there is overwhelming experimental evidence confirming the protein nature of Enzymes*.

It is hard to imagine nowadays that as late as 1926, R. Willstätter still denied that enzymes belonged to Proteins or any other known Class of organic substances, and doubts on this matter were voiced until quite recently. These doubts stemmed from experiments that yielded enzymatically active solutions, yet failed to detect protein using standard qualitative color tests. This occurs because the Enzyme Concentration, even at a high specific activity, remains below the threshold sensitivity of chemical protein assays.

The protein nature of enzymes is evidenced by the fact that Fermentation enzymes are inactivated (lose activity) upon boiling—a phenomenon first established by L. Pasteur. Boiling induces irreversible Denaturation of the enzyme-protein, causing it to lose its inherent ability to catalyze Chemical Reactions. Similarly, proteins undergo denaturation upon boiling and lose their biological properties (antigenic, hormonal, and catalytic). Under METABOLISM/18.html">The Influence of various Physical and Chemical factors (such as UV and X-ray irradiation, ultrasound, precipitation by mineral acids, alkalis, alkaloid Reagents, heavy metal salts, etc.), enzymes undergo denaturation just like other proteins.

Upon Hydrolysis, enzymes—much like proteins—break down into Amino Acids, which undoubtedly serves as compelling proof of the protein nature of enzymes**.

Interesting data pointing to the protein nature of enzymes were obtained in the laboratory of I.P. Pavlov. When determining the digestive capacity of gastric juice, a direct correlation was discovered between this capacity and the protein content of the juice. Consequently, it was concluded that Pepsin in gastric juice is a protein.

Compelling proof of the protein nature of an enzyme includes obtaining it in a pure state and isolating it in the form of protein crystals. To date, over 1,000 crystalline enzymes have been obtained. The Structure of many of them has been studied in detail using modern Methods of Protein chemistry and molecular physics [such as X-ray crystallography, nuclear magnetic Resonance (NMR), and electron paramagnetic resonance (EPR)].

Like all proteins, enzymes exhibit A number of properties characteristic of macromolecular compounds: amphotericity (they can exist in solution as anions, cations, and zwitterions); electrophoretic mobility due to the presence of positive and negative charges, whereas at their isoelectric point they show no mobility in an electric field. Enzymes are incapable of dialysis through semipermeable membranes, a property that allows their solutions to be freed from low-molecular-weight impurities. Like proteins, they are readily precipitated from aqueous solutions at low temperatures via salting-out or by the careful addition of acetone, ethanol, and other substances without losing their catalytic properties.

* The sole exception to this rule is the discovery of enzymatic activity in certain RNA precursor molecules, known as ribozymes, which catalyze self-splicing—i.e., the excision of non-translated intron sequences from an RNA precursor (see Chapter 13).

** Some enzymes contain a non-protein component In addition to the protein part, thus forming a conjugated protein molecule (see below).

Like proteins, enzymes have a large molecular weight—ranging from tens of thousands to several millions (Table 4.1).

Table 4.1. Molecular Weight of enzymes

Enzymes exert a highly specific action, which further proves their protein nature, given that proteins are exceptionally specific from an immunological standpoint. Finally, direct proof of the protein nature of enzymes was provided by the laboratory Synthesis of the first enzyme, ribonuclease, accomplished in 1969 in B. Merrifield's laboratory in New York*.

This automated solid-phase synthesis consisted of the sequential incorporation of all 124 amino acid residues in strict accordance with the Amino Acid Sequence (Primary Structure) of the natural pancreatic enzyme—ribonuclease **.

The artificially synthesized enzyme did not differ from natural ribonuclease in chemical, catalytic, and immunological tests.

Taking the aforementioned circumstances into account, when isolating enzymes in a pure state and storing them, one must consider a crucial property of proteins, namely stability, which is determined by a number of factors. A general rule when working with enzymes is maintaining the optimal Temperature, usually corresponding to body temperature, or using a temperature near 0°C for preparative purposes.

* The complete Introduction/19.html">Primary structure of pancreatic ribonuclease was deciphered in 1955 by S. Moore and W. Stein.

** The artificial synthesis of a second enzyme, Lysozyme, consisting of 118 amino acid residues, has also been successfully carried out.

It should be kept in mind, however, that a few enzymes are highly sensitive to low temperatures; notably, the mitochondrial enzyme (ATPase) that catalyzes ATP breakdown undergoes inactivation at 0°C while remaining stable at room temperature. Most enzymes retain their stability at pH 6.0–8.0, although exceptions exist. For preparative purposes, enzymes are frequently dehydrated (Water removal) in a vacuum from a frozen solution (a method known as "lyophilization"). Precipitation of enzymes from solution using alcohol or acetone is also performed at low temperatures, since these Procedures lead to an almost total loss of enzymatic activity at room temperature. To stabilize an enzyme, chelating agents are often employed: for example, ethylenediaminetetraacetate (EDTA) is added to the enzyme:

EDTA can bind undesirable impurities (trace amounts of heavy Metal Ions such as copper, lead, mercury, etc., present in reagents) that inhibit enzyme activity. An essential prerequisite for maintaining enzyme stability is storing them in a dried or frozen state (under cold conditions). Many enzymes are stable as Suspensions in concentrated ammonium sulfate solutions.