CHEM30111 Lecture 1 Handouts - Organic Cofactors PDF

CHEM30111, Lecture 1: Overview & Introduction Organic Green cofactors chemistry applications What are they? Bio- Wha...

CHEM30111, Lecture 1: Overview & Introduction Organic Green cofactors chemistry applications What are they? Bio- What is their inspired/ role? biomimetic How do they catalysis function? Electron density for enzyme bound cofactor PLP and substrate 1 Why should we care? 2  Syllabus & ILOs Definition and overview of organic cofactors (Lect 1) Covalent catalysis by PLP/TPP (Lect 2-3) Workshop (Lect 4) Redox catalysis by FAD/FMN/NAD(P)H (Lect 5-6) Workshop (Lect 7) ILO1 – Describe the role of organic cofactors in biocatalysis and identify various cofactors based on their structure ILO2 – Explain how the structure of organic cofactors is tailored to the corresponding biocatalytic function ILO3 – Evaluate the effect(s) on protein binding and/or biocatalytic function of cofactor structure modifications ILO4 – Rationalise the component steps in mechanisms of covalent catalysis by PLP/TPP and highlight aspects under enzyme control ILO5 – Rationalise the component steps in redox mechanisms catalysed by FAD/FMN/NAD(P)H and highlight aspects under enzyme control 3  Enzymes, proteins & amino acids Substantial rate enhancement, often >> 105 Regio- and stereo-selective Operate under narrow window of ambient conditions (i.e. pH, temperature, solvent etc) Activity often highly dependent on conditions Relatively narrow substrate specificity Vast majority is protein based 4  Enzymes, proteins & amino acids Proteins are polypeptides Majority has a defined 3D-structure linked to function Structure only stable under a narrow window of ambient conditions (i.e. pH, temperature, solvent etc) Sequence governs structure/function 20 amino acids, average protein ~300 amino acids 20300 sequences!!! 5  Enzymes, proteins & amino acids Proteins are flexible Considerable flexibility in the structure, often also linked to (catalytic) function Sequence governs structure/function and thus also the dynamic behaviour Recap on proteins: https://www.youtube.com/watch? time_continue=3&v=wvTv8TqWC48 6  Enzymes, proteins & amino acids 20 amino acids Diverse set of chemical functionalities But limited to acid/base or weak nucleophiles s nucleophilie Weak No electrophiles Little scope for redox chemistry (exception is disulphide formation), or radical chemistry Hence for ~50% of enzymes: Recruitment of additional chemical electrophiles Weak functionality through (non)-covalent binding of (in)organic cofactors/metal ions or, on more rare occasions, chemical modification of amino acids. Binding/cofactor specificity is property of protein structure/sequence Enzyme will exploit synergy between 7  Cofactors, coenzymes & prostetic groups Definition of cofactors: any non-protein substance required for an enzyme to be catalytically active. Some cofactors are inorganic, such as metal ions in various oxidation states. Others, such as most vitamins, are organic molecules. Cofactors are either bound tightly or may be loosely associated with the enzyme. They may also be important for structural integrity, i.e. in their absence the enzyme does not Organic fold properly Inorganic or becomes unstable. In/Organic A 4Fe4S cluster, bound by 4 Cys A heme cofactor, consisting of a The flavin cofactor FAD residues central Fe bound by a tetrapyrrol 8 ligand  Cofactors, coenzymes & prostetic groups Hence, not all cofactors are directly involved in catalytic mechanism! To exclude a pure allosteric mode of action, coenzymes are defined as cofactors that must be present in the active (i.e. substrate binding) site of an enzyme. Active Example: site/Substrate binding site prFMN K+ Metal binding Mn2+ site Activity is prFMN dependent, Activity is Mn2+ and K+ and this cofactor is dependent, but neither responsible for the key involved in chemistry, role is chemistry, so this is a to aid in prFMN binding, so coenzyme in this case 9 cofactors, but not  Cofactors, coenzymes & prostetic groups We can also further differentiate between coenzymes, which bind to an enzyme at the beginning of a catalytic cycle and leave the active site afterwards, and prosthetic groups, which are tightly bound within an enzyme and remain there throughout many reaction cycles. Examples: prFMN K+ Metal binding Mn2+ site In this system, the FMN flavin cycles The prFMN coenzyme between two protein components, remains bound throughout per total reaction cycle, and is thus a multiple cycles: prosthetic coenzyme for the luciferase but not a group  Organic cofactors: overview A complete overview of organic cofactors: Too many to cover in one 7 lecture unit!!! We will mainly focus on 4 cofactors : PLP, TPP, the flavins FMN and FAD and NAD(P)H 11  Organic cofactors: overview Focus on 4 cofactors : PLP, TPP, the flavins FMN/FAD and NAD(P)H PLP (pyridoxal phosphate) FMNH2 (flavin mononucleoti de) TPP (thiamine pyrophosphate) NAD+ (nicotinamide Anything these have in common? dinucleotide 12  Organic cofactors: overview Focus on 4 cofactors : PLP, TPP, the flavins FMN/FAD and NAD(P)H PLP (pyridoxal phosphate) FMNH2 (flavin mononucleoti de) TPP (thiamine pyrophosphate) NAD+ (nicotinamide Why so large/complex? dinucleotide 13  Organic cofactors: overview Focus on 4 cofactors : PLP, TPP, the flavins FMN/FAD and NAD(P)H PLP (pyridoxal phosphate) FMNH2 (flavin mononucleoti de) TPP (thiamine pyrophosphate) NAD+ (nicotinamide Can you highlight the most reactive dinucleotide 14 positions? 15  Organic cofactors: overview phosphate groups appear in all, most also have aromatic systems (ie largely 2D) PLP (pyridoxal phosphate) FMNH2 (flavin mononucleoti de TPP (thiamine pyrophosphate) NAD+ (nicotinamide dinucleotide 16  Organic cofactors: overview Active groups only form part of (much) larger entities… why? PLP (pyridoxal phosphate) FMNH2 (flavin mononucleoti de TPP (thiamine pyrophosphate) NAD+ (nicotinamide The most reactive positions highlighted dinucleotide 17 in blue  Analogy: Cofactors as tools X Acts on Y using tool Business end/actual tool Handle Tool specific Handle! 18  Organic cofactors Structure is linked to activity Group transfer, Redox active/group Redox active, amine substrates transfer hydride transfer PLP (pyridoxal phosphate) FMN (flavin mononucleoti de TPP (thiamine Grouppyrophosphate) transfer, NAD+ (nicotinamide carbonyl substrates dinucleotide 19  Many cofactors are vitamins Redox active, Group transfer, Redox active/group hydride transfer, amine substrates, transfer, Vit B2 Vit B3 Vit B6 Vitamins are inactive precursors, requiring modification to create suitable “handle” and or activate the reactive group. Group transfer, carbonyl substrates, Vit B1 20  Cofactor binding and activity control sit tive Ac e 21  Cofactor binding and activity control Adaptive radiation of finches (Darwin): beak shape adapts to food source Enzymes evolve too: grey is cofactor binding, red area controls substrate binding and chemistry. 22  Cofactor binding and activity control Enzymes often evolve as modular entities: red region has become more diversified, leading to diverse chemistry when considering a family of enzymes. Ie the active site 23 adapts to the substrate  Studying Cofactor binding proteins: Spectroscopy, Structural biology UV-Vis spectroscopy allows direct monitoring of chemical state of most cofactors Why only possible to observe/study above 300nm? 24 25 Cofactor versus protein Protein absorbs < 300nm! Similar problems with 1D NMR, IR, protein effectively shields most spectral features from the cofactor 26  Studying Cofactor binding proteins: Spectroscopy, Structural biology Working well are UV-Vis (> 300nm); mass spectrometry, fluorescence, EPR 27  Studying Cofactor binding proteins: Spectroscopy, Structural biology Often in conjunctio n with Mechanistic crystal interpretati spectrosco on py 28  Studying Cofactor binding proteins: Spectroscopy, Structural biology 29  Recap Organic cofactors What are they? Small organic molecules (many also vitamins) bound by enzymes What is their role? Provide additional key chemical functionality required for function/catalysis How do they function? Electron density for enzyme bound cofactor PLP and substrate: The synergy of an Protein able to control enzyme–cofactor system enables a functional level that could not be activity and ensures achieved by the cofactor or the enzyme 30 Next lecture: PLP chemistry 31  Any questions? 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