Synthesis and Turnover Enzyme 2024 PDF

Synthesis and Turnover Enzyme Dr. Yekti Asih Purwestri Biochemistry laboratory, Faculty of Biology Universitas Gadjah Mada  As we remember ! Most enzymes are proteins so Mechanism of enzyme synthesis is no different from protein synthesis in general The informati...

Synthesis and Turnover Enzyme Dr. Yekti Asih Purwestri Biochemistry laboratory, Faculty of Biology Universitas Gadjah Mada  As we remember ! Most enzymes are proteins so Mechanism of enzyme synthesis is no different from protein synthesis in general The information which determines the primary sequence of an enzyme is contained in the order of DNA sequence Enzymes are apparently synthesized not singly but as part of a sequence of the enzymes required for the successive steps in a metabolic pathway. A series of structural genes determines the molecular composition of the enzymes. From these genes, molecules of messenger RNA (ribonucleic acid) carry the transcribed list of instructions into the cell cytoplasm, where the ribosomes of the rough surfaced reticulum, with the assistance of transfer RNA assemble the individual amino acids into the required enzyme molecule. Posttranslational Processing of Proteins Folding Amino acid modification (some proteins) Proteolytic cleavage FOLDING Before a newly translated polypeptide can be active, it must be folded into the proper 3-D structure and it may have to associate with other subunits. Enzymes/protein involve in folding process 1. Cis-trans isomerase for proline → Proline is the only amino acid in proteins → forms peptide bonds in which the trans isomer is only slightly favored (4 to 1 versus 1000 to 1 for other residues). Thus, during folding, there is a significant chance that the wrong proline isomer will form first. Cells have enzymes to catalyze the cis-trans isomerization necessary to speed correct folding. 2. disulfide bond making enzymes 3. Chaperonins (molecular chaperones) → a protein to help keep it properly folded and non-aggregated. a. Some proteins capable to fold into its proper 3-D structure by itself without any help of other molecules b. Some proteins need chaperones to fold (example in human hsp 70) c. Some proteins need bigger protein → chaperonins to be able to fold correctly. Chaperones → Function to keep a newly synthesized protein from either improperly folding or aggregating After synthesized, protein needs to fold in order to have its function The folding pattern is dictated in the amino acid sequence of the protein. Chaperonins → a polysubunit protein form “a cage” like shape → give micro environment to protein Protein Targeting Nascent proteins → contain signal sequence → determine their ultimate destination. Bacteria → newly synthesized protein can: stay in the cytosol, send to the plasma membran, outer membrane, periplasmic, extracellular. Eukaryotes → can direct proteins to internal sites → lysosomes, mitochondria etc. Nascent polypeptide → E.R and glycosylated → golgi complex and modified → sorted for delivery to lysosomes, secretory vesicle and plasma membrane.  Translocation → The protein to be translocated (called a pro-protein) is complexed in the cytoplasm with a chaperone The complex keeps the protein from folding prematurely, which would prevent it from passing through the secretory porean ATPase that helps drive the translocation after the pro-protein is translocated, the leader peptide is cleaved by a membrane-bound protease and the protein can fold into its active 3-d form. Signal recognition particle (SRP) detects signal sequence and brings ribosome to the ER membrane Control of enzyme biosynthesis In living cell → not all enzymes are synthesized with maximum velocity all the time. The rate of enzymes production → controlled in accordance with metabolic need state of development of the cell The main point in the control of enzyme synthesis → copying of the genes of the DNA in the form of mRNA  A → B The rates of formation of enzyme which are controlled by repressor → regulated by the metabolic state of the cell [A] → too high → induction by substrate [B] → too high → repression by product Repressor does not by itself bind to the operator → has specific binding site for the product So repressor-product → bind to the operator Example → amino acid synthesis Synthesis of bacterial enzymes Jacob and Monod (1961) coordination and control of enzyme synthesis are essential for correct cellular function and at a given moment, most of the potentialities inherent in the genome must be inactive or repressed. the rate of enzyme synthesis is under the control of regulator and operator genes, with a repressor molecule in the cell cytoplasm acting as a link between the two. There are two basic systems of control 1. Inducible → the repressor molecule is synthesized, under the coded instructions of a regulatory gene, and in its active form, it prevents the formation of specific proteins. Inducer will inactivate repressor 2. Repressible → the repressor molecule is considered to be active only when combined with a corepressor, and it is the absence of the corepressor which initiates new enzyme synthesis, in a process known as derepression. https://www.sciencedirect.com/topics/biochemistry-genetics- and-molecular-biology/enzyme-synthesis#  Enzyme Induction usual process for catabolic enzyme sequences Axample: tyrosine as the inducing substrate for a series of enzymes required to convert it to noradrenalin. Increasing concentration of tyrosine induces increased concentration of enzymes for its conversion. When all available tyrosine is converted, repressor molecules are free to attach to the operator gene once more, and inhibit enzyme synthesis. Enzyme Repression usual process for anabolic enzyme sequences Corepressors are generally the final products of the enzyme sequence Example: synthesis of tyrosine, one of the many amino-acids in insulin. If there is sufficient tyrosine in the synthesizing cell, then the enzymes for its synthesis are unnecessary, and the genes coding for its synthesis are repressed by the active repressor/tyrosine complex. If the tyrosine is absent, the repressor, unaided by its corepressor, becomes inactive, and allows transcription of the genes coding for the enzymes needed for tyrosine synthesis.  Enzyme induction and derepression both involve new synthesis of protein, and both are inhibited by drugs which block protein synthesis, such as actinomycin D. In contrast, activation of a preformed enzyme is not affected by such drugs. Both induction and repression of enzymes are usually highly specific. Inducers are generally the specific substrates for enzymic action or their analogues. Repressors are generally the products of the enzymic action, or their analogues. Repression of an enzyme denotes inhibition of its synthesis, not of its activity. Feedback inhibition, on the other hand, denotes inhibition of the activity of the first enzyme in a series, by the end—product of the biosynthetic chain which it initiates. Enzyme turn over Proteins are targeted for destruction Proteins have different half-lives Most enzymes that are important in metabolic regulation have short lives Also important for removal of abnormal proteins / enzymes Proteolytic enzymes are found through out the cell  Several proteases → present in the eukaryotic cytosol two Ca2+ activated proteases → calpains an ATP-dependent protease → proteasome Four structural features are currently thought to be determinants of turnover rate : 1. Ubiquitination 2. Oxidation of amino acid residues 3. PEST sequences 4. N-terminal amino acid residue A small protein present in all eukaryotic cells ubiquitin → tagging protein for destruction Three enzymes participate in the conjugation of ubiquitin to proteins 1. Terminal carboxyl of ubiquitin link to a sulfhydril group of E1 2. Activated ubiquitin then shuttled to a sulfhydril of E2 3. Target protein is tagged by ubiquitin for degradation 4. Ubiquitin-specific protease recognize the target → degrade 2. Oxidation of amino acid residues Conditions that generate oxygen radicals cause many proteins to undergo mixed-function oxidation of particular residues Conditions require Fe2+ and hydroxyl radical, and the amino acids most susceptible to oxidation are → lysine, arginine, and proline. E. coli and rat liver contain → protease → cleaves oxidized glutamine synthetase in vitro, but does not attack the native enzyme accumulation of oxidatively damaged protein beyond the cell’s capacity to degrade and replace them contribute to importantly to cellular aging 3. PEST sequence all short-lived proteins (i.e., half-lives < 2 h) → contain 1 or more regions rich in proline, glutamate, serine, and threonine 4. N-terminal amino acid residue An N-terminal protein residue of Phe, Leu, Tyr, Trp, Lys, or Arg → short metabolic lifetimes →Proteins with other termini are far longer-lived. Thus, the intracellular half-life of a particular protein depends on the identity of its N-terminal amino acid residue. Video Pendukung Pembelajaran Repressible and Inducible Operons https://www.youtube.com/watch?v=v5VWcDdf0JA Operon https://www.youtube.com/watch?v=10YWgqmAEsQ Ubiquitin Proteasome System Programme https://www.youtube.com/watch?v=hvNJ3yWZQbE