Proteins

1) 2) 3) 4) 5) 6) 7) 8) Chemistry Protein Structure Enzymes Iso-Enzymes Amino Acid Metabolism Urea Cycle Metabolism of C Skeleton and AA Vitamins in Metabolism

1) CHEMISTRY OF PROTEIN

Alpha carbon is optically active. Asymmetrical except in glycine Naturally occurs in alpha form(exception- mono Na-glutamate - aginomoto) Physiological pH R group polar / nonpolar -NON POLAR AA y Glycine glucose y Valine - vodinam y Isoleucine insuline y Leucine - -line y Proline protein y Tryptophan trytcaranne y Methionine metformin y Phenylalanine peththa y Alanine allala Proline

S Containing AA - Methionine and Cysteine

Isoelectric pH

In isoelectric pH

Net charge is zero Solubility is minimum (precipitation occurs) No movement in the electric field Zwitter ion predominates cation and anion are equal in proportion

Pi

ka1+ ka2

At pK weak acids and bases(conjugated) exist in equal amount.

Recognize AA AA + Ninhydrine

Blue-purple colour

Qualitative measure can be done but not quantitative measure using ninhydrin. Exception Glycine pink Praline yellow Histidine green Used to detect aminoaciduria

Protein structure
Peptic bond- partial double bond features. Covalent bond. AA AA 25< polypeptide 25> oligopeptide

1. 2. 3. 4.

Primary Secondary Tertiary Quaternary

1. Primary structure Determine the physical and chemical properties Not destroyed in protein denaturation 2. Secondary structure (eg- keratin) Only responsible H-bonds 1. Intra molecular 2. Inter molecular - helix Intra chain h-bonds Right handed helixes (n=4)

- pleated sheet Parallel Anti parallel

H-bonds-

Interchain and intrachain perpendicular to the axis. turns are important to bevel secondary structures.

Tertiary structure Globular- Enzyme,myoglobin Fibrous-collagen,elastin Ionic,disulphide,hydrophobic,hydrophilic bonds are responsible. One or more than one secondary structures combined. End product is fibrous or globular protein. Myoglobin Skeletal muscle,heart muscle Function- O2 storage Primary structure 153 AA Secondary structure 8- -helix- A to H Tertiary structure- globular Interior non polar AA - hydrophobic Exterior- polar AA-H bonds

Collagen- Triple helix Most abundant protein in body Water insoluble Secreted by fibroblasts as tropocollagen. Secondary structure- -helix Type 1 bones,ligaments Type 2 hyaline,elastin Type 3 Reticuline Type 4 basement membrane 3 left handed helixes Right handed twist Gly-X-Y-Gly-X-Y-Gly-X-Y X,Y- proline, hydroxyproline lysine,hydroxylysine. Need ascorbic acid in synthesis-(vit-C) Quaternary structure Should have more than one secondary structure Eg- Iso enzyme.Haemoglobin ( ) Protien denaturaion Loose biological activity- because change the 3D structure. Reversible or irreversible Due to- high temperature Chemicals Toxins Detergents

triple helix

ENZYMES Holo enzyme = Apo enzyme + Co factor a. Co-enzyme (organic) b. Metal ion(inorganic)

Prosthetic groups Co enzymes or protein tightly bound to the protein All enzymes are water soluble Enzyme specificity 1) Substrate Absolute- Glucokinase Relative- Hexokinase 2) Stereo specificity Only act on D or L isomers D-CHO- glycolytic enzymes

3) Reaction specificity Catalize only one type of reaction. Eg- protein kinase Protein phosphatase. Mechanism of action

E+S

E+S complex

E + product

1)Induced fit Can describe only the relative substrate specificity. 2)Lock and key Only for absolute substrate specificity.

Km can differ due to temperature , pH,structure of substrate. Km is independent from enzyme concentration

Enzyme activation

Inactive enzyme Trimming (.) Trimming endoprotease Inactive Enzyme Trypsinogen enterokinase Metal ions

active enzyme

Active Enzyme Trypsin

(.) Metal Ions - Cl- - in salivary amylase - Mg2+ - kinases

Enzyme Inactivation 1) Competitive 2) Non competitive 3) Uncompetitive

1- Competitive Inhibition -Competitive with substarate -Competitive for the active site -Inhibitor is structurally similar to the substrate -Which has high concentration binds with the active site -Reversible

Eg:- HMG Coa reductase - statin drugs Succinate DH - malanate Acetylcholine esterase neostigmine PABA analogue sulfer drug Methotrexate folic acid Not non competitive reversible

2) Non competitive inhibition -Inhibitor bind to both enzyme & enzyme substrate complex -Cant overcome by adding extra substrate

Uncompetitive inhibitor -Inhibitor binds at the site other than active site -Cant overcome by adding extra substrate

Irreversible Inhibition Metal ions, CN-, Organophosphates, Methotrexate, Penicilline.

Allosteric Enzyme

Effecter binds constantly & alters the affinity for substrate. (-)ve effecter inhibits enzyme activity (+)ve effecter increases enzyme activity Iso Enzyme - Are physical distinct molecular forms of the same enzyme - Has different site of synthesis - But catalyzes the same reaction Because of - Different genes - Different post transitional modifications - Different subunits Oligomeric more than one subunit Homomulyimer all subunits are same Heteromultimer subunits differ Lactate DH (LDH) (5) LDH1, LDH2 Heart, RBC LDH3 Brain LDH4, LDH5- Liver, Skeltal LDH2, LDH3 increases in acute Leukemia, LDH3 increases in malignancies and LDH4, LDH5 increases in Liver damage -Blood LDH2> LDH1 -Heart LDH1>LDH2

Alkaline Phosphatase (ALP) Alpha1-ALP- Bile, Epithelial tissue Alpha2- ALP- Heat labile liver Heat stable placenta

Pre beta ALP bone Gamma ALP intestine Leukocyte ALP

Acid Phosphatase (ACP) Tatrate liable prostate non tatrate liable

prosthetic carcinoma non prostate

Creatine Kinase (CK) CK-BB brain CK-MBheart CK-MMmuscle Gamma Glutamyl Transferase (GGT) GGT increases in Alcohol abuse, cholestasis(bile obstruction)

Metabolism of AMINO ACIDS

Catabolism of AMINO ACIDS

AA

Non essential Ala,Asn,Asp,Tyr,Cys,Glu, Gln,Gly,Pro,Ser

essential Lie,Leu,Lys,Thr,Trp,Met, Phe,Val Arg,His(semi essential)

Diet

Catabolism

Body protein

AA pool

synthesis of nitrogenous compounds

Synthesis (NEAA)

protien synthesis

y y y

Free AA in all body fluid >50% in skeletal muscle plasma

Bio synthesis of non essential AA

1.

AST/GOT glutamate oxaloasetate transaminase Rich in heart muscles ALT/GPT - glutamate pyruvate transaminase Rich in liver

2. Glu , Gln synthesized by assimilation of free NH3

Glu.DH.

KG

NH3

Glu

H2O

NADH/NADPH

NAD/NADP

Glu + NH3 Mg-ATP

Gln synthase

Gln Mg-ADP

3. Others  KG

Glu

Proline

Ornithine

Arginine

 3 phosphoglyserate

3 phosphohydroxy pyruvate AA KA

Glycin

serine

phosphoserine

Methyline H4F

H4F

ATP  Asp + Gln

AMP Asn + Glu

Asn synthase

 Met

S adenosyl methionine serine Cystathione

Cystine Phenyl alanine hydroilase  Phenyl alanine Structure of glutamate Thyrosine

Gln Synthase

Catabolism of Amino acids

Excess amino acid not stored degradade

Fat C skeleton Energy Glucose Conversion of Alpha-Amino groups to Urea

Transmaniation transdeamination Oxidative deamination Ammonia metabolism Urea cycle

Transamination Alpha amino group of a amino acid is transferred to a alpha-ketoacid. Enzymes Transaminases or Amino transferases Requires VitB6 (pyridoxal phosphate) co-enzyme

Transaminases are both cytosolic and mitochondric

Importance of transmaination 1- Synthesis of non essential amino acids 2- Channel alpha aminogroups of amino acids to Glu, because Glutamate is the only AA that can undergo appreciable rate of transamination in mammalian tissue 3- Provides C-skeleton for energy. Glucose, ketoacids, FA

Oxidative deamniation 90% Liberate NH3 - reversible Glutamate

glutamate dehydrogenase

KG NADH/NADPH

NH3

NAD/NADP

Amino acid oxidase -10% Irreversible Co enzyme flevo protein

What are the sources of ammonia inside our body? - Deamination of AA - Hydrolysis of glutamine eg- kidneus

- By gut flora Normal NH3 level 6 15 micro mol per litre

Disposal - Amination of ketoacids - Biosynthesis of pyrimidines - Biosynthesis of glutamine (Nh3 is very toxic to the brain tissue and eliminated, requires ATP) - Urea synthesis

Alanine Glucose cycle Transport of amino - nitrogen

Urea Cycle

Important things about urea cycle - Two molecules of NH3 ( gets it from Nh3 and Aspartate) - Requires 3 ATP molecules for the synthesis of urea molecule - CO2 and Fumarate from TCA cycle - Aspartate from OA

Regulation 1- CPS I - N-acetyl glutamate is allosteric activator synthesized analogue carbomyl, glutamate 2- Altering urea cycle enzymes in liver eg- excessive saturation increases

Metabolic disorders Clinical Symptoms Vomiting in infancy, avoidance of high protein foods, intermittent ataxia, irritability, lethargy, drowsiness, mental retardation, tremor, slurring of speech, blurring of vision, hepatic coma. Disorders 123456-

Hyperammonemia type-1 - deficiency of CPS-I Hyperammonemia type-2 - deficiency of ornithine transcarbomylase Citrullinemia - deficiency of arginosuccinate synthase Arginosuccinic acid urea - deficiency of arginosuccinase Hyperargininemia - deficiency arginase N-acetyl glutamate synthase deficiency treat with analog- carbomyl glutamate

Also delay in maturation of urea cycle enzymes and deficiencies and blood bypass liver, liver cell dysfunctions causing hyperammonemia.

Hepatic Coma Due to increased NH3 in minute volume causing toxicity of the brain. Elimination needs ATP - depletion of ATP ATP Glu + NH3 ADP Gln
Gln synthase

Treatment 1. Benzoate Benzoate + Co A Benzyl Co A Glycine Hippurate

Hippurate excreted in urine

2.

Phenyl acetate Phenyl acetyl glutamine

Phenil acetate + Gln 3. Neomycin oral antibiotic 4. Lactulose

Metabolism of Carbon skeleton

Ketogenic

Acetoacetate , Acetyl Co A , Acetoacetyl Co A

End products
Glucogenic Pyruvate or intermediate of TCA cycle

  

Leu -

Purely ketogenic

Ketogenic and glucogenic - Ile , Phe ,Try , Trp , Lyc Others are glucogenic

CO2 Glu GABA

Phenylketouria - Deficiency of phenyl alanine hydroxylase - Accumulates Phe in body fluid - Metabolized in to phenylketones and excreted in urine - Symptoms - irritability, tremors, convulsions - Increased Phe inhibits tyrosinase, causes impaired melanin synthesis - Treatment - restriction of Phe in diet

Albinism - Defect in tyrosine metabolism - Deficiency in the production of melanin - Can be partial or total absence of the pigmentation of the skin, hair or eyes - It can be autosomal recessive, dominant, or x-linked - Complete absence of pigments in skin, hair and eyes is called occulocuataneous albinism - Due to the deficiency of Tyrosinase - Lack of melanin in the skin photo sensitivity - Lack of melanin in the eyes - photophobia Cystinuria - Excretes dibasic AA in urine - C-cys - A-arg - L-lys - 0-ornithine - Due to the defect in tubular reabsorption - Cause to renal stones - Give penicillamine

Alcaptonuria - Deficiency of homogentisate oxidase - Accumulation and excretion of homogentistic acid - Dark colour urine arthritis

Hartnup disease - Decreased absorption and increased excretion of Tryptophan - Low NAD coenzyme - Pellagra like symptoms

Maples syrup urine disease - Defective decarboxylation of branched chain ketoacid - Increased basic chain ketoacid in blood and urine - BCCA Ile , Val , Leu

Homocystinuria - Increased Met, Low Cys - Homocystine and SAM excreted in urine - In protein - homocyteine is incorporated instead of cystein - Brittle hair