Enzymes and Kinetics

Enzymes are biological catalysts — almost all proteins — that accelerate chemical reactions by lowering the activation energy (Ea) without being consumed. Without enzymes, most biochemical reactions would proceed too slowly to sustain life. Understanding enzyme function and kinetics is fundamental to pharmacology, diagnostics, and metabolic medicine.

Active Site and Substrate Binding

The active site is a three-dimensional cleft or pocket formed by amino acid residues that may be distant in primary sequence but brought together by protein folding. Substrate binding depends on complementarity of shape, charge, and hydrophobicity between substrate and active site. The lock-and-key model (Fischer, 1894) proposed a rigid complementary fit; the induced-fit model (Koshland, 1958) — now supported by structural evidence — proposes that substrate binding induces a conformational change that optimally positions catalytic residues around the substrate.

Catalytic Mechanisms and Transition State Stabilisation

Enzymes stabilise the transition state more tightly than substrate or product, reducing Ea. Key mechanisms include: acid-base catalysis (histidine at pH 7 acts as proton donor/acceptor), covalent catalysis (transient enzyme-substrate covalent intermediate, e.g., serine proteases), metal ion catalysis (Zn2+ in carbonic anhydrase activates water), and proximity/orientation effects (binding substrates in the optimal geometry for reaction). Serine proteases (trypsin, chymotrypsin, elastase) use a catalytic triad of serine, histidine, and aspartate to cleave peptide bonds via an acyl-enzyme intermediate.

Michaelis-Menten Kinetics

The Michaelis-Menten equation describes the relationship between reaction velocity (v) and substrate concentration [S]: v = Vmax[S] / (Km + [S]). Vmax is the maximum velocity when all active sites are saturated with substrate. Km (the Michaelis constant) is the substrate concentration at half-maximal velocity; it approximates enzyme-substrate affinity — a lower Km means higher affinity. The turnover number kcat (= Vmax / [E]total) is the number of substrate molecules converted per active site per second. The specificity constant kcat/Km measures catalytic efficiency. A Lineweaver-Burk (double-reciprocal) plot of 1/v vs 1/[S] gives a straight line with slope Km/Vmax, y-intercept 1/Vmax, and x-intercept −1/Km, and is used to determine kinetic constants and identify inhibitor types graphically.

Enzyme Inhibition

Competitive inhibitors resemble the substrate and bind reversibly to the active site; they increase apparent Km without changing Vmax and are overcome by excess substrate. On a Lineweaver-Burk plot, competitive inhibition changes the x-intercept but not the y-intercept. Uncompetitive inhibitors bind only the enzyme-substrate complex, decreasing both apparent Vmax and apparent Km. Non-competitive (mixed) inhibitors bind both free enzyme and the ES complex, decreasing Vmax without changing Km. Irreversible inhibitors form covalent bonds with the active site: aspirin irreversibly acetylates cyclooxygenase (COX), and organophosphate nerve agents phosphorylate acetylcholinesterase. Allosteric regulation involves effector binding at a site distinct from the active site, causing conformational changes that alter activity — the basis of metabolic feedback loops.

Enzyme Regulation and Zymogens

Enzyme activity is regulated by covalent modification (phosphorylation/dephosphorylation by kinases and phosphatases), allosteric effectors, and zymogen activation. Zymogens are inactive enzyme precursors activated by proteolytic cleavage: trypsinogen → trypsin (by enteropeptidase), prothrombin → thrombin. This prevents autodigestion of the synthesising tissue and allows precise spatial and temporal activation.

Clinical Diagnostics: Plasma Enzyme Assays

Measuring plasma enzyme activities is a cornerstone of clinical diagnosis. In myocardial infarction, cardiac troponin I (cTnI) is the gold-standard biomarker, rising within 3–6 hours and remaining elevated for 7–10 days. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are released from damaged hepatocytes; an AST:ALT ratio >2:1 suggests alcoholic hepatitis. Lipase and amylase are elevated in acute pancreatitis. Alkaline phosphatase (ALP) elevation occurs in cholestatic liver disease or bone disorders. Understanding enzyme kinetics and isoforms allows clinicians to interpret these assays accurately.