Clinical Chem Enzymology

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

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