Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19UNIT III:AMINO ACIDS, PROTEINS, & ENZYMESLecture 4: Regulation of Enzyme ActivityWhy do we need to regulate enzymes??It is critical that living organisms be able to quickly and efficiently respond to changes in their environment. This is true for individual cells and the living organism as a whole. - metabolism is dynamic - feeding vs fasting - changes in diet or activity levels - dividing vs quiescent cells - pathophysiological insults (diseases, injury, infection, &c) - countless other examplesIt is inefficient to synthesize new enzyme (protein) when we need increased enzyme activity and/or destroy enzyme when we need decreased activity (although this is necessary or best in some cases)It is usually more efficient to modulate the activity of preexisting enzyme molecules: - activating them when increased activity is needed - turning them off when decreased activity is required.How do we regulate enzyme activity??Regulation at the “gene expression” level – how much mRNA is made for a given enzyme, and how efficiently mRNA is translated into new protein - transcriptional and translational control – more in Biochem 108!“Post-translational regulation – regulation of enzyme activity after the enzyme has been synthesized - zymogen activation (activation of preexisting inactive enzyme) - reversible covalent modification – “on/off” switch (phosphorylation/dephosphorylation most common) - allosteric effectors – regulatory molecules affect enzyme activity by binding at sites other than the active site. They can be activators or inhibitors. - inhibitors that bind to the active site, altering enzyme activityControlled degradation (total destruction) of enzyme molecules - relatively rare - sometimes very important (e.g., proteins involved in cell cycle/cell division) - again, more in Biochem 108!“Genetic” regulation of enzyme activityGenes are regions of DNA that contain the DNA sequence for synthesis of a specific protein, many of which are enzymesTranscription of a gene into mRNA is controlled by regulatory proteins(transcription factors) that bind to specific sequences of DNA and stimulate or inhibit transcription of the gene into mRNA. mRNA is translated into new proteins by ribosomes.Binding of these regulatory proteins to DNA is, in turn, regulated by other regulatory molecules.Lots more about this in Biochem 108 next semester!new proteinControlling enzyme activity by controlling levels of the enzyme - synthesis of new enzyme protein - degradation of existing enzymeis a very crude and metabolically expensive approachGood for long-term regulation (days/months/years) - dietary changes - growth/maturation/aging Most required regulation of enzyme activity is relatively short-term (seconds/minutes/hours)It involves modifying the activity of preexisting enzymeMost regulation of enzyme activity is “short-term”Regulation of enzyme activity: zymogen activationSome enzymes are synthesized as “zymogens” - inactive precursors of the active form - contain extra amino acids that prevent them from being catalytically active - activation involves proteolysis (removal of part of the peptide chain) - removal allows proper folding for active enzyme44 aa segmentblocking activesitePepsinogen(inactive)Pepsin(active)freeactivesiteproteolysis44 aapepsin(active protease)pepsinogenblocksactivesiteZymogen activation goodwhen you need lots ofactive enzyme very quicklyThe pancreas synthesizes inactive proteases . These zymogens are released into the duodenum when partially digested food enters from the stomach.Enteropeptidase, a highly specific protease, cleaves one of these zymogens, trypsinogen, to active trypsin.Many digestive enzymes are synthesized as zymogensTrypsin in turn activates the remaininginactive proteases by selectiveproteolysis. These active proteasesdigest dietary proteins.This zymogen activation sequence is an example of a biochemical cascade,where a small initial event triggersa rapid and large response.Zymogens can be recognizedby the prefix “pro” or thesuffix “ogen” added to the name of the enzymeProcarboxypeptidase carboxypeptidase Proelastase elastaseChymotrypsinogen chymotrypsinPreproinsulin insulinBiochemical cascades – rapid amplification of a small signalA single enzyme molecule repeats the same Rx many times (very rapidly too!)Enzymes are very efficient at amplifying signals within cells or accomplishing other required amplificationsBlood clotting and complement activation (infection fighting) are other examples of vital zymogen activation cascades (Biochem 108)Metabolic control by zymogen activation is expensive – enzymes must be degraded to stop activity (NOT a problem for digestive enzymes) - but sometimes the best way to do things11,0001,000,000Regulation of enzyme activity by reversible covalentmodification: phosphorylation / dephosphorylationPhosphorylation: - occurs at specific serine, threonine, or tyrosine (-OH) residues on a protein - is carried out by protein kinases (ATP required) - is reversed by protein phosphatasesProtein phosphorylation is controlled by specific signals - hormones (e.g., insulin, glucagon) are major players - VERY rapid (sec/min)Addition or removal of the very polar & (-)charged phosphate group changes the protein’s configuration. This shape change alters its activity - substrate binding &/or catalytic activity – can be (+) or (-)VerilyImportant!Allosteric Regulation of Enzyme ActivityAllosteric (another shape – or site!!) regulation of enzyme activity involves reversible non-covalent binding of a small molecule to an enzyme Binding of the allosteric regulatory molecule causes a change in the shape of the enzyme, altering its activity. This can be a positive or negative effect.Allosteric enzymes have multiple subunits (quaternary structure). Changes in one subunit result in changes in the other subunits.Allosteric regulation by a substrate is a special case – it is exactly analogous to binding of oxygen to hemoglobin (remember??)SS SSSSSSSS SS= inactive (low affinity)= active (high affinity)Subunit conformationsAllosteric regulation by a substrate is almost always positive (activation)Allosteric Regulation of Enzyme Activity by “Effectors”Many multi-subunit