Learning Outcome

• To understand the clinically important enzymes and isoenzymes, as well as their changes in certain disorders.

Enzyme

• Biological catalyst that increases the rate of a biochemical reaction

  • catalyses the conversion of substrates → products

  • specific

• Not undergoing permanent changes or being consumed

• Alter the rate, NOT the equilibrium point

Enzyme properties

• Most are proteins or conjugated proteins

• primary structure - linear sequence of the amino acids

• secondary structure - three-dimensionally coiled polypeptides

• tertiary structure - subsequent folding of secondary structure

• Active site

  • interaction and binding site for substrate

  • a cleft or groove formed by the specific folding pattern of specific amino acid residues

    • determines substrate specificity

Enzyme reaction conditions

Temperature

• optimal temperature for most enzymes: below 40C

• doubling of enzymatic activity with an increase of 10C

pH

• change in pH affects the ionisation state of acidic or basic amino acids

  • change the structure and shape of enzyme → affect/inactivate enzyme activity

• each enzyme has an optimal pH value, ranging from pH 1.5-8.0

Enzyme concentration

• ↑enzyme concentration

  • ↑ active sites to convert substrate molecules into products

  • needs to have an excess of substrate

• zero-order kinetics

  • a reaction under the condition of substrate excess

  • all active sites of enzymes are saturated in an excess of substrate

  • rate of reaction us independent of substrate concentration

  • concentration of product formed over a specified period of time depends on the enzyme concentration

Substrate concentration

• ↑ substrate concentration, ↑ rate of product formation, at a constant enzyme concentration

• rate of product formation is linear & proportional to the substrate concentration, until a limiting point is reached

• limiting point

  • all active sites are occupied with substrates

  • rate of reaction at its maximum

  • further increase in the substrate concentration does not increase the reaction rate

Enzyme Nomenclature

• Name if the substrate/group targeted by the enzyme, followed by the suffix -ase

  • e.g., urease → enzyme that hydrolyses urea

• Name indicated the reaction catalysed by the enzyme

  • e.g., glucose oxidase enzyme that catalyses the oxidation of glucose to gluconolactone & hydrogen peroxidase

• Empirical name, e.g., trypsin & pepsin

Alkaline Phosphate

• Membrane-bound glycoprotein on the outer layer of cell membrane

• exists as a group of isoenzymes in different tissue sites

• found on membranes and cell surfaces of small intestine, kidneys, liver, bone & placenta

• majority (>80%) of alkaline phosphatase in serum is liver isoenzyme, followed by the bone isoenzyme & a small amount from intestine

• Physiological functions:

  • catalyses the hydrolysis of phosphate monoesters at alkaline pH

  • releases inorganic phosphate from the substrate

  • requires divalent cations as cofactors

  • only found in the tissues under normal physiological condition

  • increased production under specific condition may release it into circulation

Clinical Relevance

Liver

• significantly increased activity in obstructive hepatobiliary disease & osteoblast-mediated bone disease

  • elevated serum alkaline phosphatase level

• biliary tract obstruction: 3-10x the upper limit of reference interval

• hepatobiliary disorders: alkaline phosphatase + other markers of hepatic function

Bone (bone isoenzyme)

• elevated serum level is common in osteoblast-related disorders

Cancer

• primary or metastatic cancer with local bile duct obstruction & increasing leakage of liver isoenzyme

Pregnancy (placental isoenzyme)

• elevated serum level starting 16 weeks of gestation

• 2-3x the upper limit of the reference interval in third trimester

Decreased level

• less common

• hypophosphatasia, postmenopausal women receiving oestrogen therapy for osteoporosis, Wilson’s disease, magnesium deficiency, hypothyroidism & severe anaemia

Lactate Dehydrogenase

• An oxidoreductase important in anaerobic metabolic pathway

• present in almost all tissue, but significant level in heart, skeletal muscle, liver & kidneys

• higher concentration in the cytoplasm than serum

• tetramers of either of the 2 types of subunits:

  • muscle (M)

  • heart (H)

• Exhibits 5 isomeric forms:

  • LDH1 - 4 heart subunits; heart tissue

  • LDH2 - 3 heart & 1 muscle subunits; reticuloendothelial system

  • LDH3 - 2 heart and 2 muscle subunits; lungs

  • LDH4 - 1 heart and 3 muscle subunits; kidneys

  • LDH5 - 4 muscle subunits; liver & skeletal muscle

• Physiological functions:

  • involved in the anaerobic metabolism of glucose when there is an absence or limited supply of oxygen in tissues

  • catalyses the reversible conversion of pyruvate to lactate, with the use of NADH

  • lactate is transported to the liver

    • reversibly converting lactate to pyruvate

• Clinical relevance:

  • slight to moderate elevation: acute myocardial infarction, pulmonary embolism, leukaemia, haemolytic anaemia, liver & renal disease

  • significantly elevated: pernicious anaemia, megaloblastic anaemia, certain cancers