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3-dimensional structure: monomer oligomer Variants:1) isozymes (different genes)- tissue-specific forms
2) allozymes (different alleles at single genetic locus)
3) post-translational modifications- cell and tissue specific forms,
E.g., liver- and bone-specific alkaline phosphatase (ALP) differ only in carbohydrate contents attached to the ALP proteins.
Enzymes: properties and measurements (II)
Measurements:
1) kinetic properties and activity of enzyme [S], [P], Km, v, Vmax
2) expression of activity measurements
1 unit of enzyme activity (U) = 1 micromol per min.
Then, U/l of sample or U/ml of sample (e.g.,plasma, serum, blood, urine etc.) are widely used.
3) direct measurement of enzyme protein concentration. This is limited due to very low concentrations of proteins.
I. Clinical Correlations of Enzymes: Principles of Diagnostic Enzymology
1. Introduction: A human cell contains 28,602 different proteins, 2,709 proteins of which are enzymes. They are assigned roles in ~ 135 metabolic pathways (2,645 metabolites)
They are distributed in: cytosol, nucleus, rough and smooth ER, Golgi bodies, mitochondria, lysosomes, plasma and organellar membranes. The enzymes are mainly synthesized in cytosol or ER (stay, or target to other organelles and export to extracellular environment, or extracellular
enzymes). A relatively small numbers are synthesized in the mitochondria and these enzymes stay within this space.
The activity of an intracellular enzyme is determined by the rates of synthesis, inactivation and degradation (= turnover).
Figure 1. Turnover of intracellular enzymes
2004Nobel Prize Chemistry : CCCCCCCCCCC, ,
Figure 2. Mechanism of enzyme release from damaged cell
Table 1. Half-lives of clinically important enzymes in plasma Enzyme Range (hours) Lactate dehydrogenase (LD) LD-1 (H4) 50-70 LD-5 (M4) 8-14 Alanine transaminase (ALT, GPT) 40-50 Aspartate transaminase (AST, GOT) mitochondrial AST 6-7 cytosolic AST 12-17 Creatine kinase (CK) CK-MM 10-20 CK-MB 7-17 CK-BB 3 Alkaline phosphatase (ALP) liver ALP 190-230 bone ALP 30-50
Most enzymes are present in cells at much higher concentrations than in plasma. Some occur predominantly in cells of certain tissues. Normal plasma enzyme level (normal or reference range, e.g., 5’-nucleotidase, ALP, amylase =2-15, 30-95, 95-290 U/L). The normal levels reflect the balance between the rate of synthesis and release into plasma during cell turnover, and the rate of clearance from the circulation. The enzyme level in plasma may be:•increased due to proliferation of cells, an increase in rate of cell turnover or damage or in enzyme synthesis, or to reduced clearance from plasma;•lower than normal, due to reduced synthesis, congenital deficiency.
Table 2. Serum normal (reference) ranges of clinical enzymes
Enzyme Abbreviation Range Stability (male> female)
2. Assessment of cell damage and proliferation:•Changes in plasma enzyme levels may help to detect and localize tissue cell damage or proliferation, or to monitor treatment and progress of disease.
•Plasma enzyme levels depend on: a) the rate of release from damaged cells which, in turn, depends on the rate at which damage is occurring; b) the extent of cell damage.
•In the absence of cell damage, the rate of release depends on: a) the rate of cell proliferation; b) the degree of induction of enzyme synthesis.
•These factors are balanced by : a) the rate of enzyme clearance from the circulation (only partly known); b) half-life of enzyme.
Introduction: Isozymes are enzymes catalyzing the same catalytic reaction, but they are synthesized from different genes/loci, and most contain subunits (quaternary structure). This will result in cell and tissue-specific forms of isozymes.Examples of some clinically significant isozymes are as follows:
A.1. HIV-1 Protease: HIV-1 has an aspartate protease, which is essential for delivery of structural and functional processing of the gag and gag-pol viral gene products.
Protease inhibtor design, with known3-D structure at active site complexed with the drug with a Ki of 0.25 nM
Figure 4.
A.2. HIV-1 Reverse Transcriptase (RT):RT is a viral polymerase responsible for synthesis of viral DNA strand.The RT inhibitors are majority for drugs in clinical use.
- - 103 1K L mutation > HIV re -13sisttodr ugs
Crystal of drug 4Figure 5.
A.3. Avian flu, H5N1 virus: Neuraminidase (NA) Enzyme NA is responsible for release of virion from the host epithelial cells by cleaving the receptor sialic acid. NA inhibitor is the drug in clinical use, e.g., zanamivir (Relenza) and oseltamivir (Tamiflu)
CCCCCCC CC CC-CCCCCCC complex Figure6. CCCCCCCC CCCCCC C f Tami fl u on vi r al NA
Tamiflu
B). Alzheimer’s disease (AD)B.1. AD involves mutation of gene APP (chromosome 21) coding for amyloid beta protein, this APP deposits in neurons of brain as amyloid fibrils -->and leads to cell damage.Loss of cholinergic cell is accompanied by reduced : - ChAT(choline acetyltransferase), ACh (acetylcholine), - AChE (acetylcholine estease).
This is compensated byincreased BuChE(butyrylcholinesterase) from Glial cell.
Rational design is to inhibit the BuChE, ACh is not destroyed.
-ve
Figure 7.
-ve
Fig 8.
Abnormal or mutant APP (amyloid beta protein) deposits in neurons as amyloid fibrils --> cell damage.
B.2. Design of drug for treatment of Alzheimer’s disease : Cholinesterase as enzyme target, phenothiazine derivatives as drug.
Figure 9.
V.Enzymes abnormalities in metabolisms
1. Excess enzyme activity
Gout is characterized by elevated uric acid levels in blood and urine, due to overproduction of de novo purine nucleotides.
then leads to increase degradation of purines to uric acid through xanthine oxidase.
Figure 10.
[PRPP] + ve
2. Enzyme deficiency
Identification and treatment of enzyme deficiency. Enzyme deficiencies usually lead to increased accumulation of specific metabolites in plasma and hence in urine. This is useful in pinpointing enzyme defects. E.g., De novo pyrimidine pathway: defects of OPRT and OMPDC leads to accumulation of orotate ----> Hereditary orotic aciduria (Gene mapping, 3q13; inheritance pattern, autosomal recessive).