Unit 5 Question Bank
Enzymes are additionally stereospecific, typically catalysing the reaction of only one stereoisomer — D- but not L-sugars, L- but not D-amino acids. Stereospecificity arises from binding at at least three points of attachment, which fixes the substrate's orientation so that apparently identical groups become distinguishable.
Significance: specificity is what allows a cell to conduct and independently control a broad spectrum of biochemical processes simultaneously in the same small volume.Harper's ch.7, p.61 · TMU Lecture 5 Slides 10, 12
Characteristics:
• It comprises only a small part of the molecule.
• It is a three-dimensional entity: the residues are far apart in the primary structure and are brought together by folding.
• It is usually located in a cleft or crevice, shielding the substrate from water.
• It provides a distinct microenvironment, whose polarity, hydrophobicity, acidity or alkalinity may differ markedly from the surrounding cytoplasm.
• Substrate binding involves specific non-covalent interactions, bringing substrates into close proximity and optimal alignment with the cofactors, prosthetic groups and side chains that catalyse the change.Harper's ch.7, pp.62–63 · TMU Lecture 5 Slide 30
Prosthetic group — tightly and stably incorporated into the protein's structure by covalent or non-covalent forces. Examples: pyridoxal phosphate, FMN, FAD, thiamin pyrophosphate, biotin. Metal ions are the commonest type; enzymes with tightly bound Fe, Co, Cu, Mg, Mn or Zn — about a third of all enzymes — are metalloenzymes.
Cofactor — associates reversibly and transiently with the enzyme or with the substrate, and therefore must be present in the surrounding medium for catalysis to occur. Most are metal ions; such enzymes are metal-activated enzymes.
Coenzyme — a recyclable shuttle transporting substrates from one point in the cell to another. Its role is twofold: to stabilise reactive species such as hydride ions (NADH) or hydrogen atoms (FADH), and to act as a handle facilitating recognition of small groups such as acetate (CoA). Many are derivatives of B vitamins.Harper's ch.7, p.62 · TMU Lecture 5 Slides 14–15, 30
Although the reaction is identical, their physical, chemical and immunological properties differ significantly — which is what allows them to be separated by electrophoresis. Differences may include sensitivity to regulatory factors or substrate affinity (hexokinase and glucokinase), adapting them to particular tissues or circumstances; they also provide a “backup” copy of an essential enzyme.
Example: lactate dehydrogenase is a tetramer of H (heart) and M (muscle) subunits, giving five isozymes. LDH-1 predominates in heart and rises in myocardial infarction; LDH-5 predominates in liver and rises in liver disease.Harper's ch.7, pp.66, 69 · TMU Lecture 5 Slides 23–25
Substrate and enzyme induce mutual conformational changes that facilitate both substrate recognition and catalysis. It replaced Emil Fischer's “lock and key” model, which pictured a rigid complementary site and failed to account for the dynamic changes that accompany catalysis.Harper's ch.7, pp.62–64
Proteins that diverged from a common ancestor and share a large number of conserved residues are said to be homologous. Among the most highly conserved residues are those that participate directly in catalysis — evolution tolerates change almost anywhere except the active site.Harper's ch.7, p.66 · TMU Lecture 5 Slide 22
Definition
The active site is the three-dimensional catalytic centre of an enzyme — a cleft or pocket, usually at the surface, formed by the aminoacyl residues that bind the substrate and carry out catalysis. It is where the enzyme-substrate (ES) complex forms and where the chemical transformation takes place.
Characteristics
| Characteristic | Explanation |
|---|---|
| It occupies only a small part of the molecule | Relatively few residues form the site; the remainder of the protein positions them correctly and provides the structural scaffold |
| It is a three-dimensional entity | The catalytic residues are typically far apart in the primary structure and are brought into proximity only by the folding of the polypeptide. This is why tertiary structure is indispensable to function |
| It lies in a cleft or crevice | The substrate is bound in a pocket that shields it from water |
| It provides a distinct microenvironment | Its polarity, hydrophobicity, acidity or alkalinity may differ markedly from that of the surrounding cytoplasm, and this environment itself contributes to catalysis |
| Binding involves specific interactions | Substrate is held non-covalently by hydrogen bonds, ionic and hydrophobic interactions, conferring the enzyme's specificity |
| It aligns the reactants optimally | Substrates are brought into close proximity to one another and into optimal alignment with the cofactors, prosthetic groups and side chains that catalyse the transformation |
How the substrate is accommodated
Emil Fischer first likened the specificity of the site to a lock accepting only the proper key, having observed that bound substrate makes an enzyme more resistant to thermal denaturation. However, the rigid lock-and-key picture failed to account for the dynamic changes that accompany catalysis. Daniel Koshland's induced fit model corrected this: substrate binding induces a conformational change in the enzyme, and enzyme and substrate change each other's shape — a hand entering a glove. This flexibility is essential, because a site that closes around its substrate can strain the bond destined for cleavage.
The general principle
Enzymes enhance reaction rates by factors of 10⁶ or more, and they do so by lowering the activation energy of the reaction — not by altering ΔG or the position of equilibrium. Harper's identifies four general mechanisms, used in combination.
1 · Catalysis by proximity
For molecules to interact they must come within bond-forming distance. By binding substrates at its active site, an enzyme creates a region of high local substrate concentration in which the molecules are held in the ideal orientation to react. This alone produces rate enhancements of at least a thousandfold.
2 · Acid-base catalysis
The ionisable functional groups of aminoacyl side chains — and of prosthetic groups where present — contribute to catalysis by acting as general acids (proton donors) or general bases (proton acceptors). Specific acid or base catalysis involves only protons or hydroxide ions; general acid or base catalysis responds to all the acids or bases present.
Example — HIV protease, an aspartic protease. Aspartate X acts as a base, abstracting a proton to activate a water molecule; the activated water attacks the peptide bond, forming a transient tetrahedral intermediate; Aspartate Y then acts as an acid, donating a proton to the newly formed amino group and facilitating breakdown of the intermediate. Shuttling the proton from Asp X to Asp Y restores the enzyme.
3 · Catalysis by strain
Enzymes that catalyse lytic reactions bind their substrate in a conformation that is unfavourable for the bond targeted for cleavage. This strained conformation mimics the transition state, selectively stretching and distorting the bond, weakening it and rendering it more vulnerable. Linus Pauling was the first to propose transition-state stabilisation as a general mechanism of enzyme catalysis; the idea is exploited to design transition state analogues as drugs.
4 · Covalent catalysis
The formation of a covalent bond between the enzyme and one or more substrates, generating a transient covalent intermediate. It often follows a “ping-pong” mechanism, in which the first substrate binds and its product is released before the second substrate binds. Chymotrypsin and fructose-2,6-bisphosphatase illustrate covalent catalysis.
A supporting point
The residues that participate directly in catalysis are among the most highly conserved in any enzyme family, and enzyme and substrate induce mutual conformational changes in one another that facilitate both recognition and catalysis.
Definition
Isozymes are distinct enzyme forms that catalyse the same reaction. That is the only property they share: although the reaction is identical, their physical, chemical and immunological properties differ significantly.
Origin
Like other protein families, isozymes arise through gene duplication. A second copy of a gene is created, and the two copies then evolve independently. Unlike the divergent homologs described for the proteases — where chymotrypsin and trypsin acquired different substrate specificities — isozymes retain the same reaction but acquire subtle differences in properties such as sensitivity to regulatory factors or substrate affinity (hexokinase and glucokinase are the classic pair). These differences adapt them to specific tissues or circumstances. Isozymes may also enhance survival by providing a “backup” copy of an essential enzyme.
Lactate dehydrogenase — the worked example
LDH catalyses lactate + NAD⁺ → pyruvate + NADH + H⁺. It is a tetrameric enzyme assembled from two monomer types — H (for heart), encoded by LDHB, and M (for muscle), encoded by LDHA. Four subunits drawn from two types give five possible tetramers:
| Isozyme | Composition | Predominates in |
|---|---|---|
| LDH-1 | HHHH | Heart |
| LDH-2 | HHHM | |
| LDH-3 | HHMM | |
| LDH-4 | HMMM | |
| LDH-5 | MMMM | Liver |
The relative proportion of each subunit in a given organ is determined by tissue-specific patterns of expression of the H and M genes. LDH therefore acquires tissue specificity as a direct consequence of its quaternary structure.
Clinical importance
In health, LDH exists mainly within cells. In disease, damaged cells release their own characteristic isozyme pattern into the serum, so that the tissue of origin can be inferred from the pattern of LDH isozymes in plasma.
- Myocardial infarction → elevated LDH-1.
- Liver disease → elevated LDH-5.
Because their physical properties differ, individual isozymes are readily separated by electrophoresis and detected using a coupled assay. Detection is possible at all because of amplification: each enzyme molecule transforms thousands of substrate molecules, and under appropriate conditions the rate of reaction is proportional to the amount of enzyme present. For NAD(P)⁺-dependent dehydrogenases such as LDH the assay is spectrophotometric — NADH and NADPH absorb at 340 nm whereas the oxidised forms do not, so the rate of change of absorbance reports enzyme quantity.
The wider diagnostic picture
LDH is one of a family of non-functional plasma enzymes used diagnostically: AST and ALT (myocardial infarction and viral hepatitis), amylase and lipase (acute pancreatitis), creatine kinase (muscle disorders and MI), alkaline phosphatase (bone and obstructive liver disease), ceruloplasmin (Wilson disease). Many are not specific to the disease listed.
The history of cardiac markers illustrates the principle that an enzyme appearing in plasma only after 12 hours or more is of limited utility. LDH and the aminotransferases were superseded by creatine kinase — whose isozyme CK-MB appears 4–6 h after infarction, peaks at 24 h and returns to baseline by 48–72 h — and CK has in turn been replaced in most laboratories by cardiac troponins I and T, which rise within 2–6 h and remain elevated for 4–10 days.