Amino Acids, Proteins & Enzyme Catalysis

Amino acids are the fundamental building blocks of proteins, and a thorough understanding of their structure and chemistry is essential for appreciating protein function and enzyme catalysis in biological systems.

Amino Acid Structure and the Twenty Standard Amino Acids

Every standard amino acid shares a common backbone: a central alpha-carbon bonded to an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom, and a distinctive side chain (R-group) that defines the identity and properties of each amino acid. At physiological pH (~7.4), the amino group is protonated (-NH3+) and the carboxyl group is deprotonated (-COO-), making amino acids exist as zwitterions. The twenty standard amino acids are divided into categories based on their R-groups: nonpolar/aliphatic (glycine, alanine, valine, leucine, isoleucine, proline, methionine), aromatic (phenylalanine, tyrosine, tryptophan), polar uncharged (serine, threonine, cysteine, asparagine, glutamine), positively charged at pH 7.4 (lysine, arginine, histidine), and negatively charged at pH 7.4 (aspartate, glutamate).

pKa Values and Titration Curves

The pKa of the alpha-amino group is typically 9-10, while the alpha-carboxyl group has a pKa of approximately 2. Side chains with ionisable groups have their own pKa values: histidine imidazole ~6.0, cysteine thiol ~8.3, lysine epsilon-amino ~10.5, arginine guanidinium ~12.5, aspartate and glutamate side chains ~3.7-4.3, and tyrosine phenol ~10.5. A titration curve for a simple amino acid such as alanine shows two buffering regions corresponding to the two pKa values, with the isoelectric point (pI) midway between the two. For amino acids with ionisable side chains, the pI is calculated from the two pKa values flanking the zwitterionic form.

Peptide Bonds and Protein Primary Structure

Amino acids are linked by peptide bonds -- covalent bonds formed between the carboxyl group of one amino acid and the amino group of the next, with the loss of water (condensation reaction). The peptide bond has partial double-bond character due to resonance, making it planar and rigid, and it exists predominantly in the trans configuration to minimise steric clashes. A chain of amino acids linked by peptide bonds is a polypeptide. The sequence of amino acids from the N-terminus to the C-terminus constitutes the primary structure, which is encoded by the gene and determines all higher-order structure.

Secondary Structure: Alpha-Helices and Beta-Sheets

Secondary structure arises from hydrogen bonding between backbone amide (N-H) and carbonyl (C=O) groups. In an alpha-helix, the backbone coils into a right-handed helix with 3.6 residues per turn; each backbone C=O hydrogen-bonds to the N-H four residues ahead. Side chains project outward. In a beta-sheet, extended polypeptide strands align laterally, with hydrogen bonds between strands. Strands can run parallel (same N-to-C direction) or antiparallel (opposite directions); antiparallel beta-sheets form stronger, more linear hydrogen bonds. Proline, with its cyclic structure and lack of a backbone N-H, acts as a helix breaker and frequently appears at turns and loops.

Tertiary and Quaternary Structure

Tertiary structure is the overall three-dimensional fold of a single polypeptide chain, stabilised by hydrophobic interactions (burial of nonpolar side chains in the protein interior), hydrogen bonds, ionic interactions (salt bridges), van der Waals forces, and disulfide bonds (covalent S-S bonds between cysteine residues). Quaternary structure describes assemblies of two or more polypeptide subunits, held together by noncovalent forces and occasionally disulfide bonds. Haemoglobin (two alpha and two beta subunits) is a classic example, exhibiting cooperative oxygen binding that depends on quaternary interactions.

Protein Folding and Molecular Chaperones

Protein folding is driven thermodynamically by the hydrophobic effect -- burying nonpolar residues away from water decreases the entropic cost imposed on surrounding water molecules. The native (correctly folded) state represents the global free-energy minimum for physiological conditions. Molecular chaperones (e.g., Hsp70, GroEL/GroES) prevent inappropriate aggregation of newly synthesised or stress-denatured proteins by binding exposed hydrophobic regions and providing a sequestered environment for folding. Misfolded proteins are targeted for degradation by the ubiquitin-proteasome system. Failure of proteostasis underlies diseases such as Alzheimer's (amyloid-beta aggregation), Parkinson's (alpha-synuclein), and cystic fibrosis (misfolded CFTR).

Enzyme Catalysis: Active Site and Substrate Binding

Enzymes are biological catalysts -- almost always proteins -- that accelerate chemical reactions by lowering the activation energy without being consumed. 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 tertiary folding. Substrate binding is governed by complementarity of shape, charge, and hydrophobicity between substrate and active site. The induced-fit model (Koshland) proposes that substrate binding induces a conformational change that optimally positions catalytic residues around the substrate, contrasting with the older lock-and-key model.

Transition State Stabilisation and Catalytic Mechanisms

Enzymes stabilise the transition state of a reaction more tightly than either substrate or product, reducing the activation energy. Catalytic mechanisms include: acid-base catalysis (histidine is a common proton donor/acceptor at pH 7), covalent catalysis (formation of a 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) employ a catalytic triad of serine, histidine, and aspartate to cleave peptide bonds via a covalent 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 enzyme active sites are saturated; Km (the Michaelis constant) is the substrate concentration at half-maximal velocity and approximates enzyme-substrate affinity (lower Km = 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; for diffusion-limited enzymes it approaches 108-109 M-1s-1. 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.

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. Uncompetitive inhibitors bind only the enzyme-substrate complex, decreasing both apparent Vmax and apparent Km. Non-competitive (mixed) inhibitors bind both free enzyme and enzyme-substrate complex, decreasing Vmax without changing Km. Irreversible inhibitors form covalent bonds with the active site (e.g., aspirin acetylates COX; organophosphates phosphorylate acetylcholinesterase). Allosteric regulation involves binding of effector molecules at sites distinct from the active site, causing conformational changes that alter activity -- fundamental to metabolic regulation.

Clinical Applications: Enzyme Assays

Measuring plasma enzyme activities is a cornerstone of clinical diagnosis. In myocardial infarction, cardiac troponin is the gold-standard biomarker, but creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) isoforms also rise and fall with characteristic time courses. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are released from damaged hepatocytes; elevated ALT is relatively liver-specific, while AST elevation with a high AST:ALT ratio (>2:1) suggests alcoholic hepatitis. Alkaline phosphatase (ALP) elevation points to cholestatic liver disease or bone disorders. Amylase and lipase are elevated in acute pancreatitis. Understanding enzyme kinetics and isoforms allows clinicians to interpret these assays accurately in context.