Can an enzyme have multiple substrates?

Coenzymes

Some enzymes are active without coenzymes. However many, require a coenzyme to be active. An enzyme that is inactive in the absence of its coenzyme is called an apoenzyme. In the presence of its coenzyme to produce the active form of the enzyme, it is called a holoenzyme:

apoenzyme+coenzyme⇌holoenzyme

Although some enzymes contain a coenzyme that is tightly bound, others may contain a coenzyme that is readily dissociable. In the latter case, the coenzyme can be considered as a reactant or substrate. Thus, in the lactate dehydrogenase reaction, for example, pyruvate and the coenzyme NADH need to be added to the enzyme and, kinetically, this would be considered to be a two-substrate reaction:

pyruvate+NADH⇌lactate+NAD+

In a double reciprocal plot in which 1/velocity (y-axis) is plotted against 1/[pyruvate] holding the concentration of NADH high, the plot would give the Km for pyruvate. A similar experiment in which [pyruvate] was at a saturating level and [NADH] was varied, the reciprocal plot would give the Km for NADH.

In general, coenzymes are vitamins or derivatives of vitamins. They are listed in Table 5.4.

Table 5.4. The Vitamins, Their Coenzymes, and Their Chemical Functions

VitaminCoenzymeReaction CatalyzedHuman Deficiency DiseaseWater-Soluble VitaminsNiacin (niacinate)NAD+, NADP'OxidationPellagraNADH, NADPHReductionRiboflavin (vitamin B2)FAD, FMNOxidationSkin inflammationFADH2, FMNH2ReductionThiamine (vitamin B1)Thiamine pyrophosphate (TPP)Two-carbon transferBeriberiLipoic acid (lipoate)LipoateOxidation–DihydrolipoateReductionPantothenic acid (pantothenate)Coenzyme A (CoASH)Acyl transfer–Biotin (vitamin H)BiotinCarboxylation–Pyridoxine (vitamin B6)Pyridoxal phosphate (Pl.P)DecarboxylationAnemiaTransaminationRacemizationCα–Cβ bond cleavageα,β Eliminationβ-SubstitutionVitamin B12Coenzyme B12IsomerizationPernicious anemiaFolic acid (folate)Tetrahydrofolate (THF)One-carbon transferMegaloblastic anemiaAscorbic acid (vitamin C)–ScurvyWater-Insoluble (Lipid-Soluble) VitaminsVitamin A–––Vitamin D––RicketsVitamin E––Vitamin KVitamin KH3Carboxylation

This table is reproduced from http://wps.purshall.com/wps/media/objects/724/741576/InstructorResources/Chapter_25/Text%20Images/FC25_TB01.JPG.

Reproduced from G. Litwack, Human Biochemistry and Disease, Academic Press/Elsevier, Table 3-1, page 119, 2008

Lipoproteins, Apolipoproteins, Receptors, and Processing Enzymes

Lipoproteins are complex macromolecular structures coated by a water-compatible envelope of phospholipids, free cholesterol, and apolipoproteins covering a hydrophobic core of cholesteryl esters and triglycerides. Lipoproteins vary in size, density in the aqueous environment of plasma, and lipid and apolipoprotein content (Fig. 48.2andTable 48.1). The classification of lipoproteins reflects their density in plasma (the density of plasma is 1.006 g/mL) as gauged by flotation in an ultracentrifuge. The triglyceride-rich lipoproteins (TRLs) consist of chylomicrons, chylomicron remnants, and very-low-density lipoprotein (VLDL) and have a density of less than 1.006 g/mL. The rest (bottom fraction) of the ultracentrifuged plasma consists of low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein(a) (Lp[a]).

Apolipoproteins have four major roles: (1) assembly and secretion of the lipoprotein (apo A-I, B100, and B48), (2) structural integrity of the lipoprotein (apo B, E, A-I, and A-II), (3) coactivators or inhibitors of enzymes (apo A-I, A-V, C-I, C-II, and C-III), and (4) binding or docking to specific receptors and proteins for cellular uptake of the entire particle or selective uptake of a lipid component (apo A-I, B100, and E) (Table 48.2). The role of several apolipoproteins (A-IV, A-V, D, H, J, L, and M) remains incompletely understood.

Many proteins regulate the synthesis, secretion, and metabolic fate of lipoproteins; their characterization has provided insight into molecular cellular physiology and targets for drug development (Table 48.3). Discovery of the LDL receptor (LDL-R) represented a landmark in understanding cholesterol metabolism and receptor-mediated endocytosis.12 The LDL-R regulates the entry of cholesterol into cells, and tight control mechanisms alter its expression on the cell surface, depending on intracellular cholesterol. The LDL-R belongs to a superfamily of membrane receptors that include LDL-R, VLDL-R, LDL-R–mediated peptide type 1 (LRP1; apo E receptor), LRP1B, LRP4 (MGEF7), LRP5 and LRP6 (involved in the process of bone formation), LRP8 (apo E receptor-2), and LRP9.13 LRP1, which mediates the uptake of chylomicron remnants and VLDL, preferentially recognizes apo E. LRP1 also interacts with hepatic lipase. The complex interaction between hepatocytes and the various lipoproteins containing apo E involves cell surface proteoglycans that provide scaffolding for lipolytic enzymes (lipoprotein lipase [LPL] and hepatic lipase) involved in recognition of remnant lipoproteins. Macrophages express receptors that bind modified (especially oxidized) lipoproteins. These scavenger lipoprotein receptors mediate the uptake of oxidatively modified LDL into macrophages. In contrast to the exquisitely regulated LDL-R, high cellular cholesterol content does not suppress scavenger receptors, thereby enabling intimal macrophages to accumulate abundant cholesterol, become foam cells, and form fatty streaks. Sterol accumulation in the endoplasmic reticulum (ER) may lead to cell apoptosis via the unfolded protein response.14 Endothelial cells can also take up modified lipoproteins through specific receptors such as the oxidized LDL-R LOX-1.

2.3 The Role of Cofactors in Complex Enzymes

Not all enzymes consist exclusively of protein, as does lysozyme. Many include additional chemical components or cofactors which serve as tools. Such enzymes are known as qualified enzymes and have more complicated reaction mechanisms.

Cofactors can consist of one or more inorganic ions (such as Fe3+, Mg2+, Mn2+, or Zn2+) or more complex organic molecules, known as coenzymes. Some enzymes require both types of cofactors.

Coenzymes are organic compounds that bind to the active site of enzymes or near it. They modify the structure of the substrate or move electrons, protons, and chemical groups back and forth between enzyme and substrate, negotiating considerable distances within the giant enzyme molecule. When used up, they separate from the molecule.

Many coenzymes are derived from vitamin precursors, which explains why we require a constant low-level supply of certain vitamins. One of the most essential coenzymes, NAD+ (nicotinamide adenine dinucleotide), is derived from niacin. Most water-soluble vitamins of the vitamin B group act as coenzyme precursors very much like niacin.

Otto Heinrich Warburg (1883–1970, Fig. 2.3) discovered the respiratory enzyme cytochrome oxidase (Fig. 1.14) and NAD. His discovery and subsequent structural analysis was one of the shining hours of modern biochemistry. In the absence of niacin in the diet, certain enzymes (e.g., dehydrogenases) cannot work effectively in the body. The affected human will develop pellagra, a disease caused by vitamin B (niacin) deficiency. Otto Warburg developed an optical test making it possible to quantify reduced NADH at a wavelength of 340 nm (the oxidized NAD+ does not absorb light at this wavelength). It was now possible to measure essential enzyme reactions, such as the detection of glucose using glucose dehydrogenase (see Chapter: Analytical Biotechnology and the Human Genome).

Nowadays, vitamins like B2 (riboflavin), B12 (cyanocobalamin), and C (ascorbic acid) are produced by the ton using biotechnological methods (see Chapter: White Biotechnology: Cells as Synthetic Factories).

Cofactors that are covalently bonded to the enzyme are called prosthetic groups. Flavin adenine dinucleotide acts as a prosthetic group for GOD. Peroxidase and cytochrome P-450 contain a heme group, as found in myoglobin and hemoglobin. The heme group itself consists of a porphyrin ring incorporating an iron ion in its center.

Coenzymes, by contrast, have only loose bonds, and, just like substrates, they undergo changes in the binding process and are used up. Unlike substrates, however, they bind to a whole host of enzymes (e.g., NADH and NADPH of nearly all dehydrogenases) and are regenerated and recycled inside the cells (see Section 2.13). Enzymes that bind to the same coenzyme usually resemble each other in their chemical mechanisms.

While we referred to the cofactors as “tools,” the protein section of the enzyme is the “craftsman” using these tools, who is responsible for their effectiveness. As always, craftsmen and tools rely on each other to achieve the best possible result.

Classification of Enzymes in Catalysis

Enzymes can be classified systematically according to the difference between reaction and substrate specificity, and the mechanism of action. The enzyme code (EC) shows such a classification. Notation according to EC number, i.e. EC X.X.X.X, is shown as follows. The first EC number classifies the enzyme reaction mechanism into six groups, namely oxidation–reduction, transition, hydrolysis, dissociation, isomerization and synthesis (creating new chemical bonds with the initial assistance of ATP). Examples of enzymes classified by EC number are:

EC 1.X.X.X-reductase

EC 2.X.X.X-transferase

EC 3.X.X.X-hydrolase

EC 4.X.X.X-lyase

EC 5.X.X.X-isomerase

EC 6.X.X.X-ligase.

Although standards of classification differ in each group, they are subdivided by the difference between enzyme reaction and substrate specificity. These numbers are assigned to the entire enzyme, and over 3000 enzyme reactions have been assigned an EC number. Moreover, enzymes have a variety of activities; for example, ATPase catalyzes the hydrolysis of both proteins and ATP. Sometimes, the substrate that the enzyme metabolizes is omitted from the systemic naming, based on the same rules as a systemic name. For example, the systemic name of EC 1.1.1.1 is alcohol dehydrogenase. There are also many enzymes named in accordance with the naming convention, such as DNA polymerase.

Although an enzyme generally consists of protein, a few enzymes contain non-protein components such as nucleic acid. The ribozyme discovered by Thomas Cech and others in 1986 is a catalyst made of RNA, which acts on itself and cleaves RNA.

Some enzymes require other molecules to function and do not become active unless combined with cofactors (coenzyme, metal, etc.). An apoenzyme, a protein portion without a cofactor, does not have enzymatic activity, whereas a holoenzyme, a protein combined with a cofactor, has such activity.

The organic compound of the non-protein which assists an enzyme reaction in the active center is called a coenzyme. Since coenzymes are essential elements in the active form of the enzyme, they belong to a prosthetic group. Although they differ from typical prosthetic groups, they can easily separate from the enzyme and be consumed during an enzyme reaction with a substrate like NADH. For example, since the cytochrome P450 (CYP) enzyme is bound covalently with the heme iron, heme does not separate from the CYP enzyme. Therefore, this heme moiety is not called a coenzyme. Although lipoic acid is bound covalently to the enzyme, lipoic acid can be separated from the enzyme moiety, so lipoic acid is called a coenzyme. Therefore, the criteria defining coenzymes and prosthetic groups are not strict.

An enzyme may be comprised of two or more protein chains (peptide chain). When it consists of two or more peptide chains, each peptide chain is called a subunit.

Cloned Enzyme Donor Immunoassay

CEDIA is a more recent homogeneous immunoassay that uses the binding of an antibody to change the activity of genetically engineered fragments of β-galactosidase from Escherichia coli as the enzyme label. The enzyme is present as two inactive fragments, the enzyme acceptor (EA) and the enzyme donor (ED). ED contains a small portion of enzyme missing from the larger EA fragment. Antibodies that bind to the hapten that is conjugated to the ED fragment prevent the reassociating of the enzyme and reduces the enzyme activity. As the amount of drug increases, the amount of bound antibody to the ED fragment decreases resulting in an increase in enzyme activity due to the reassociation of EA and ED. The enzyme hydrolyzes chlorophenol red-β-galactoside (CPGR) to chlorophenol red (CPR) and galactoside and the enzyme activity can be measured spectrophotometrically.

3.24.2.3.1.2.2 The Dynamic Interplay of Histone Acetyltransferase Enzymes and Histone Deacetylase Enzymes in Regulating Gene Expression

HAT and HDAC enzymes antagonize each other. To elucidate exactly how the interplay between these two enzymes affects gene expression, Wang et al. (2009) used a ChIP-sequencing technique to identify the location of several HAT and HDAC enzymes throughout chromatin. Not surprisingly they found that HATs were recruited to active gene promoters and positively correlated with both histone acetylation and gene transcription. Unexpectedly, however, they found that the distribution of HDAC enzymes frequently overlapped with HATS. Thus, HDACs were recruited to active gene promoters but typically absent in the promoters of silenced genes. These data suggest that most HDACs function to reset chromatin in active gene promoters by removing the acetylation that contributed to transcription initiation. The coincident recruitment of these stop-and-go signals confers tight temporal control on transcription in response to a cell-surface signal by a rapid repression of transcription when the signal has subsided. Therefore active gene promoters cycle between acetylated and deacetylated states.

2.1 Introduction

Enzymes are the active proteins (except RNAse) that can catalyze biochemical reactions. These are biomolecules required for both syntheses as well as breakdown reactions by living organisms. All living organisms are built and maintained by these enzymes, which are truly termed as biological catalysts having the capability to convert a specific compound (as substrate) into products at higher reaction rates. Like chemical catalysts, enzymes increase the reaction rate by lowering its activation energy (Ea), hence, products are formed faster and reactions reach their equilibrium state more rapidly. The rates of most enzymatic reactions are millions of times faster than those of the uncatalyzed reactions. They can perform conversions in minutes or even in seconds which otherwise may take hundreds of years (Dalby, 2003; Otten and Quax, 2005). Enzymes are known to catalyze about 4000 biochemical reactions in living beings (Bairoch, 2000). For example, lactase is a glycoside hydrolase that is able to hydrolyze lactose (milk sugar) into its constituent galactose and glucose monomers. It is produced by various microorganisms and also in the small intestine of humans and other mammals helping to digest milk completely. Enzymes are also enantioselective catalysts, which can be used either in the separation of enantiomers from a racemic mixture or in the synthesis of chiral compounds.

Humans recognized the importance of enzymes thousands of years ago; clarification and filtration of wines and beer being the earliest examples of the application of industrial enzymes. Enzymes have been used in brewing, baking, and alcohol production since prehistoric times; however, they did not call them enzymes. One of the earliest written references to enzymes is found in Homer’s Greek epic poems dating from about 800 BC, where it was mentioned that enzymes were used in the production of cheese. The Japanese have also used naturally-occurring enzymes in the production of fermented products like sake, Japanese schnapps brewed from rice, for more than a thousand years. Some enzymes have been designed by nature to form complex molecules from simpler ones while others have been designed for breaking up complex molecules into simpler ones, also a few modify molecules. These reactions involve the making and breaking of the chemical bonds in the components. Owing to their “specificity,” a property of an enzyme that allows it to recognize a particular substrate that they are designed to target, they are useful for industrial processes and are capable of catalyzing the reaction between particular chemicals even if they are present in mixtures with many chemicals. These enzymes are environmentally safe, natural, and are applied very safely in food and even pharmaceutical industries. Still, enzymes are proteins, which like any protein can cause and have caused in the past allergic reactions, hence, protective measures are necessary in their production and applications.

Enzyme technology is an ever evolving branch of “Science and Technology.” With the intervention and influence of Biotechnology and Bioinformatics, continuously novel or improved applications of enzymes are emerging. With novel applications, the need for enzymes with improved properties are also emerging simultaneously. Development of commercial enzymes is a specialized business which is usually undertaken by companies possessing high skills in:

Screening for new and improved enzymes

Selection of microorganisms and strain improvement for qualitative and quantitative improvement

Fermentation for enzyme production

Large-scale enzyme purifications

Formulation of enzymes for sale

Enzyme technology offers industries and consumers an opportunity to replace processes using aggressive chemicals with mild and environmentally friendly enzyme processes. About 3000 enzymes are known of which only 150–170 are being exploited industrially. At present only 5% of chemical products are produced through a biological route in this green era. However, economically feasible and eco-friendly enzymatic processes are emerging as alternatives to physico-chemical and mechanical processes. Based on the different application sectors, industrial enzymes can be classified as: (1) Enzymes in the food industry, (2) Enzymes for processing aids, (3) Enzymes as industrial biocatalyst, (4) Enzymes in genetic engineering, and (5) Enzymes in cosmetics.

Today, enzymes are envisaged as the bread and butter of biotechnology because they are the main tools for several biotechnological techniques (gene restriction, ligation, and cloning, etc.), bioprocesses (fermentation and cell culture), and in analytics in human and animal therapy as medicines or as drug targets. Furthermore they find applications in several other industries, such as food and feed, textiles, effluent and waste treatment, paper, tannery, baking, brewing, dairy, pharmaceuticals, confectionary, etc. (Pandey et al., 2006).

The enzymes utilized today are also found in animals (pepsin, trypsin, pancratin, and chimosin) and plants (papain, bromelain, and ficin), but most of them are of microbial origin, such as glucoamylase, α-amylase, pectinases, etc. The advantage of using microbes for enzyme production is their higher growing abilities, higher productivity, and their easier genetic manipulation for enhanced enzyme production, etc. Enzymes produced from microbial origins are termed as microbial enzymes. Microbes are mainly exploited in industries for enzyme production. Moreover, microbial enzymes are supplied, well standardized, and marketed by several competing companies worldwide. Depending on the type of process, enzymes can be used in soluble form (animal proteases and lipases in tannery) and in immobilized form (isomerization of glucose to fructose by glucose isomerase).