Proteins and Amino Acids

Amino Acids, Proteins, and Enzyme Kinetics

Introduction

Proteins are the molecular workers of the cell, performing virtually every biochemical function: catalysing reactions (enzymes), transmitting signals (receptors, kinases), providing structural support (collagen, keratin), transporting molecules (haemoglobin, albumin), defending against pathogens (immunoglobulins), and regulating gene expression (transcription factors). Understanding their building blocks — amino acids — and the principles that govern their catalytic activity — enzyme kinetics — is essential to understanding both normal physiology and the molecular basis of disease.

Amino Acid Structure and Classification

All 20 standard amino acids share a common core: a central α-carbon bonded to an amino group (–NH₂), a carboxyl group (–COOH), a hydrogen atom, and a variable side chain (R group). At physiological pH (~7.4), the amino group is protonated (–NH₃⁺) and the carboxyl group is ionised (–COO⁻) — amino acids exist as zwitterions.

Classification by R-group chemistry:

| Class | Examples | Key Properties |

|---|---|---|

| Non-polar (hydrophobic) | Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met | Tend to cluster in protein hydrophobic cores |

| Polar uncharged | Ser, Thr, Cys, Tyr, Asn, Gln | Hydroxyl or amide groups — H-bonding |

| Positively charged (pH 7) | Lys, Arg, His | Found in active sites, DNA-binding regions |

| Negatively charged (pH 7) | Asp, Glu | Ionic interactions, metal coordination |

| Special | Cys (disulfide bonds), Pro (rigid ring — helix breaker), Gly (smallest, most flexible) | Unique structural roles |

Essential amino acids (9 in adults): His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val. Cannot be synthesised in sufficient amounts; must be obtained from diet. Deficiency causes impaired protein synthesis, growth retardation, and specific symptoms (e.g., tryptophan deficiency → pellagra-like symptoms; Lys deficiency → impaired collagen crosslinking).

Protein Structure

Primary Structure

The linear sequence of amino acids, linked by peptide bonds (amide bonds between α-carboxyl of one aa and α-amino of the next, with loss of H₂O). Peptide bonds have partial double-bond character due to resonance, making them planar and rigid — this constrains backbone conformation and is fundamental to secondary structure formation.

Secondary Structure

Local, regular repeating structural motifs stabilised by backbone hydrogen bonds:

• α-helix: right-handed helix, ~3.6 residues per turn, H-bond between C=O of residue n and N-H of residue n+4; stabilised by all backbone atoms
• β-sheet: extended strands connected by H-bonds; can be parallel (same N→C direction) or antiparallel (opposite direction — antiparallel is more stable)
• Turns and loops: non-repetitive regions connecting secondary elements; often surface-exposed and involved in ligand binding

Tertiary Structure

The overall 3D fold of a single polypeptide chain, stabilised by multiple non-covalent interactions (hydrophobic clustering — dominant driving force; H-bonds; ionic interactions; van der Waals) and covalent disulfide bonds (between Cys residues, particularly in extracellular proteins). Hydrophobic residues are buried in the protein core; hydrophilic/charged residues are surface-exposed.

Quaternary Structure

The assembly of two or more polypeptide chains (subunits). Example: haemoglobin (α₂β₂ tetramer). Enables cooperative behaviour and allosteric regulation that is impossible with monomeric proteins.

Enzyme Kinetics: The Michaelis-Menten Model

Enzymes are biological catalysts that accelerate reactions by lowering the activation energy (ΔG‡) without altering the reaction equilibrium. They are exquisitely specific (substrate specificity) and can be precisely regulated.

The Michaelis-Menten equation describes the dependence of reaction velocity (v) on substrate concentration [S]:

v = (Vmax × [S]) / (Km + [S])

• Vmax = maximum velocity when all enzyme active sites are saturated; proportional to [E]total × kcat
• Km (Michaelis constant) ≈ affinity indicator; the [S] at which v = Vmax/2; low Km = high affinity
• kcat = turnover number = catalytic cycles per enzyme per second (≈10⁶ for carbonic anhydrase)
• kcat/Km = catalytic efficiency; upper limit is diffusion-controlled (~10⁸–10⁹ M⁻¹s⁻¹)

Lineweaver-Burk Plot

Double-reciprocal plot (1/v vs 1/[S]): slope = Km/Vmax; y-intercept = 1/Vmax; x-intercept = −1/Km. Used clinically to determine inhibitor type — competitive inhibition changes slope and x-intercept; non-competitive inhibition changes y-intercept only.

Types of Inhibition

| Type | Binding Site | Effect on Km | Effect on Vmax | Example Drug |

|---|---|---|---|---|

| Competitive | Active site | ↑ (apparent) | Unchanged | Methotrexate (DHFR) |

| Non-competitive | Allosteric | Unchanged | ↓ | Cyanide (cytochrome c oxidase) |

| Uncompetitive | E-S complex only | ↓ | ↓ (equal) | NSAID effect on COX in some models |

| Irreversible | Active site (covalent) | —permanent loss— | ↓ | Aspirin (COX-1 Ser530 acetylation) |

Allosteric Regulation and Cooperativity

Allosteric enzymes have regulatory sites distinct from the active site where effectors bind and alter enzyme activity through conformational change. They often show sigmoidal (cooperative) kinetics rather than hyperbolic Michaelis-Menten kinetics — the Hill equation quantifies cooperativity (Hill coefficient n > 1 indicates positive cooperativity).

Key example: phosphofructokinase-1 (PFK-1) — the key regulated enzyme in glycolysis. Allosterically inhibited by ATP and citrate (high energy status); activated by AMP, ADP, and fructose-2,6-bisphosphate (insulin-stimulated). This matches glycolytic rate to cellular energy demand in real time.

Clinical Applications

• Enzyme-linked immunosorbent assay (ELISA): quantifies antigens by enzyme-linked antibody; absorbance proportional to analyte concentration (substrate → coloured product using horseradish peroxidase)
• Serum enzymes as biomarkers: troponin, CK-MB (myocardial injury); AST/ALT (hepatic damage); lipase (pancreatitis); ALP/GGT (cholestatic disease)
• Drugs as enzyme inhibitors: ACE inhibitors (enalapril, ramipril — competitive inhibition of ACE); statins (atorvastatin — competitive inhibition of HMG-CoA reductase); proton pump inhibitors (omeprazole — irreversible inhibition of H⁺/K⁺-ATPase in gastric parietal cells)

Summary

Amino acid chemistry determines protein structure and function from primary sequence to quaternary assembly. Enzyme kinetics — Michaelis-Menten parameters, inhibition mechanisms, and allosteric regulation — provide the quantitative framework for understanding how cells control metabolism and how drugs achieve their effects by targeting enzymes. These principles are applied daily in clinical biochemistry laboratories and in rational drug design.