Dr. Ketki K Assistant Professor Dept of Biochemistry HIMS Varanasi Nitric oxide(NO), Polyamies, Biogenic amines & Branched Chain Amino Acid Metabolism Dr. Ketki K Assistant Professor Dept of Biochemistry HIMS Varanasi

Nitric Oxide: Chemistry Metabolic fate Isoenzymes of NOS MOA Physiological action Diseases & treatment

Nitric Oxide Chemistry: Highly reactive “free radical” Half life : 3-4 second Nitric oxide synthase (NOS) reaction contains heme, FAD, FMN, NADPH & tetrahydrobiopterine Calmodulin modulates its activity The guanidino nitrogen of arginine is incorporated into NO' From the molecular oxygen, one atom is added to NO'and the other into citrulline Therefore, the enzyme is a di-oxygenase

Metabolic fate Nitric oxide : short half life (3-4 seconds) NO + O2 = NO2 → excreted in urine Very low quantity of NO: expelled through lung On exposure to superoxide anion (O2‘) nitric oxide (NO' ) is converted to a highly reactive free radical, peroxy nitrite(OONO'), which causes lipid peroxidation, cell injury and cell death

Iso-enzymes of NOS Neuronal NOS Macrophage NOS Endothelial NOS Other name nNOS iNOS (inducible) eNOS Site Central & peripheral neurons (cerebellum,GIT) Macrophages, neutrophils, hepatocytes Endothelial cells, Platlets Endocardium myocardium Location Cytoplasmic Plasma membrane Effect of calcium Activates it Does not activate it,induced by cytokines &during inflammation Gene Chromosome 12 Chromosome 17 Chromosome 7

MOA of NO In smooth muscles, NO activates guanylate cyclase ↓ Increased cGMP Activates protein kinase dephosphorylation of myosin light chain Relaxation of smooth muscles Vasodilatation

Physiological actions of NO Blood vessels: endothelial NOS (NOSe) present Potent vasodilator Deficiency of NO is a/w hypertension Excessive production of NO results in refractory hypotension, seen in patients with septicemic shock

2) In CNS, NOSn isoform is present 2) In CNS, NOSn isoform is present. Glutamate acts on NMDA receptors: longstanding calcium influx This activates NOSn. NO‘ stimulates the releasing hormones (CRH, GHRH and LHRH) Acts as neurotransmitter

3) Macrophages: isoform NOSi (i stands for inducible) 3) Macrophages: isoform NOSi (i stands for inducible). peroxy nitrite: lethal to micro-organisms(bactericidal) 4) Platelets: inhibits adhesion of platelets 5) Intestine: relaxes smooth muscles of GIT, reduces GI motility

Nitric oxide in diseases & treatment Angina pectoris: Nitroglycerin Pulmonary Hypertension: inhalation of NO Impotence :Sildenafil: PDE-5 inhibitor

Polyamines Having multiple amino groups Putrescine, spermidine and spermine (last 2 detected in human sperm/semen) Key enzyme of synthesis: ODC (short half life: 10 mt) requires PLP induced by steroid hormones

Biosynthesis SAM decarboxylase: Pyruvate as prosthetic group, only mamallian enzyme known to have bound pyruvate as prosthetic group

Degradation Excreted in urine, as conjugated form, as acetylated derivatives Oxidized to ammonia & CO2

Functions Involved in synthesis of DNA/RNA/Protein Essential for cell growth & proliferation Stabilizes membrane structure Protein kinases inhibited by polyamines

Clinical importance DFMO(difluromethyl ornithine) : inhibits polyamine synthesis in parasites useful against trypanosomes of Indian Kala azar & pneumocystis carnii parasite in AIDS 2) Excretion of polyamines : elevated in urine in all cancers eg: leukemia, ca lung,kidney ,bladder

Biogenic amines Decarboxylation of amino acids or their derivatives : forms biogenic amines

Branched chain amino acid metabolism Branched chain amino acids Valine(V): glucogenic, Leucine(L): Ketogenic Isoleucine(I): both glucogenic & ketogenic All are essential aa Provide fuel for brain during starvation

Second enzyme of the pathway: branched chain alpha keto acid dehydrogenase Complex of decarboxylase, transacylase and dihydrolipoyl dehydrogenase So, this resembles pyruvate dehydrogenase

branched chain alpha keto acid dehydrogenase Complex mitochondrial enzyme Oxidative decarboxylation 5 co-enzymes: TPP,Lipoamide,FAD,Coenzyme A,NAD+

Metabolic defects Maple syrup urine disease Intermittent branched chain ketonuria Isovaleric acidemia Hypervalinemia

Maple syrup urine disease Also c/a branched chain ketonuria Incidence: 1 in 1 lakh births Name originates from the characteristic smell of urine (similar to burnt sugar or maple sugar)due to excretion of branched chain keto acids

Basic biochemical defect : Deficient decarboxylationof branched chain keto acids(BKA) (reaction 2)

Clinical findings: Disease starts in the first week of life. characterized by Convulsions, severe mental retardation, vomiting, acidosis, coma death within the first year of life

Laboratory findings: Urine contains branched chain keto acids,valine, leucine and isoleucine. Rothera's test is positive Diagnosis depends on enzyme analysis in cells Preferably within first week of life

Treatment: Giving a diet low in branched chain amino acids Mild variant is called intermittent branched chain ketonuria This will respond to high doses of thiamine. This is because the decarboxylation requires thiamine Liver transplantation has been successfully tried in some cases of MSUD

Isovaleric acidemia: Metabolic defect: deficiency of isovaleryl CoA dehydrogenase Consequences: increased isovalerate in urine C/F: Mild Mental retardation, “cheesy” odor in breath & body fluids

Hypervalinemia: Metabolic defect: deficiency of enzyme responsible for transamination of valine alone Consequences: increased valine in plasma

Homework Aminoacids & their derivatives as neurotransmitters Table 15.7 satyanarayan