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Biostatistics and Bioinformatics
Models of Enzyme Action
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Biostatistics and Bioinformatics Models of Enzyme Action
Description of Module Subject Name Biochemistry
Paper Name 13 Biostatistics and Bioinformatics Module Name/Title 14 Models of Enzyme Action
Dr. Vijaya Khader Dr. MC Varadaraj
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1. Objectives: The learning objectives in this module are to evaluate models of enzyme action through structural analysis. This will be achieved with
1. Understanding basic steps for an enzyme catalysed reaction involving single substrate and single product.
2. Superimposing two different structures of same enzyme to achieve structural alignment.
3. Comparing active site of the same enzyme, one free active site and other active site bound to a ligand, such as substrate, transition state analog or product, to understand induced fit change in structure of active site during enzyme catalysed reaction.
4. Appreciate induced fit binding of substrate and product to a highly specific and a broadly specific enzyme.
2. Concept Map
Summary Brief Description
Computing Ligand Bound Active Site
Superimposing Structures
Induced fit binding: specific and broad specific
Binding transition state to active site Basic Enzyme Reaction
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3. Description
There are three models for understanding enzyme action. These are lock & Key, Induced fit and transition state model. In 1890, Emil Fischer proposed Lock-and-Key model for binding of the substrate to enzyme. In this model, the active site of the enzyme is complementary to substrate . In 1958, Daniel Koshland proposed induced fit model for binding of the substrate to enzyme. In this model, the active site of the enzyme is not complementary to substrate initially, but is made complementary to substrate, upon entering of the substrate in the active site, through induced change in the structure of that active site.
However, in both of these models, the active site of the enzyme is complementary to the structure of substrate, either initially or upon induced fit. Transition state model assumes complementarity of enzyme active site to Transition state of the reaction.
Experimentally, it is possible to determine the structure of enzyme having bound substrate at the active site. Therefore, the structure of the active site bound to substrate can be useful to understand binding of the substrate. The enzyme catalysed reactions are technically reversible. The empty enzyme active site can bind either substrate or product i.e. only one at a given time. It is possible experimentally, to determine the structure of enzymes having bound product at the active site. Therefore, the structure of the product bound active site can be useful to understand binding interactions with the product. In addition, enzyme structures bound to transition state analogs are also available. Therefore, these three enzyme structures bound to substrate or to transition state analog or to product will be useful for understanding transitions in active site during enzyme catalysed reaction.
The basic enzymatic reaction sequence involves four enzyme structures: free enzyme, substrate bound to active site of the enzyme, Transition state structure bound to active site of the enzyme and product bound to the active site of the enzyme. In the last module, we have seen that the active site of enzyme is present in a cleft on the surface of the enzyme. The empty active site has some residues for binding of the ligand and other residues to achieve specific reaction chemistry. To convert a substrate to product or vice versa i.e. product to substrate, the structure of the bound ligand, either substrate or product, has to pass through a transition state structure, which exists only within enzyme active site. The isolation of transition state structure bound to active site of an enzyme reaction is not possible, because transition state structure is a fleeting structure i.e. it exists for a very short time, and therefore, it cannot be isolated and its structure cannot be determined experimentally. However, transition state analogs, which are stable molecules, can bind to enzyme. Therefore, transition state analog bound to active site can be isolated and used to determine the structure of active site in the transition state. This will reveal the structure of an active site when the enzyme active site is complementary to transition state. This structural evaluation will reveal the continuity in the transformation of enzyme active site from free from to complementarity to substrate and then complementarity to transition state and finally complementarity to product.
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When, the structure of enzyme in each of the state is available, the comparisons of these structures help in understanding the structural transitions for binding of ligands. The comparison of free enzyme with bound ligands i.e. substrate or product or transition state is very useful to understand induced fit transition of active site to bind ligands.
Therefore, the learning objectives in the present module include Understanding basic steps for an enzyme reaction involving single substrate and single product. This will follow Superimposing two different structures of same enzyme to achieve structural alignment for Comparing active site of the same enzyme, one free active site and other active site bound to a ligand, such as substrate, transition state analog or product so as to Appreciate induced fit transition of active site to bind ligands.
Go to Concept Map 3.1. Basic Enzyme Reaction
Enzyme catalysis is the increase in the rate of a chemical reaction by the active site of an enzyme. The basic reaction involves a single ligand, either a substrate or a product, bound to enzyme forming ES or EP, respectively. The enzyme catalysed reactions are technically reversible, i.e. when substrate (reactant) binds to free enzyme, it is converted to product. On the other hand, when product binds to free enzyme, it is converted to substrate. This conversion is through formation of a fleeting structure, ETS i.e. active site bound to transition state structure.
In commonly used Cleland Nomenclature, this Reaction is called Uni-Uni Reaction. In each direction of the reversible reaction i.e. forward and reverse direction, there are four steps each: For the forward direction, first step involves diffusion of the substrate to the active site of the free enzyme for formation of enzyme - substrate (ES) complex. The substrate bound enzyme complex then achieves a transition state structure bound to enzyme (ETS). This ETS complex then yields an enzyme-product (EP) complex. The product then finally diffuses away from the enzyme and the free enzyme conformation is regenerated.
Similarly, for the reverse direction, first step involves diffusion of a product to the active site of the free enzyme for formation of enzyme- product complex The product bound enzyme complex then achieves a transition state structure bound to enzyme (ETS). This ETS complex then yield an enzyme- substrate (Es) complex. The substrate then diffuses away and the free enzyme conformation is regenerated.
Therefore, Uni-Uni enzyme catalysed Reaction involves eight elementary reactions, including.
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1. Binding of substrate with free Enzyme to form enzyme bound substrate and the dissociation of bound substrate from the enzyme, to yield free Enzyme
2. Transformation of substrate bound enzyme to form transition state bound Enzyme and in reverse direction, Transformation of transition state bound Enzyme, to substrate bound enzyme.
3. Transformation of transition state bound Enzyme to form product bound enzyme and in reverse direction, Transformation of product bound enzyme, to transition state bound Enzyme
4. Diffusion of product from enzyme bound product to yield free Enzyme and in reverse direction, association of product, to from enzyme bound product.
Go to Concept Map
3.2. Detecting Active Site residues of an Enzyme
In the present module, we will carry forward the example of enzyme, OSBS. We will use three structures for analysing the structure of free active site, structure of substrate bound active site and structure of product bound to active site. First, visit pdb web interface to download OSBS structure files. To search PDB, enter “o-succinylbenzoate synthase” and then limit the search in organism Tab for E. coli only. This list presents four PDB structures.
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The Second structure in the list i.e 1FHU: crystal structure analysis of OSBS from E. Coli is wild type free enzyme and will be used for induced fit binding of ligands, i.e. substrate and product. Next, 1R6W: Crystal structure of the mutant OSBS from Escherichia coli Complex with, SHCHC , the substrate. This structure will be used for binding of the substrate with active site residues. Finally, 1FHV: crystal structure analysis of OSBS from E. coli complexed with Mg2+ and the product, i.e. OSB. This structure will be used to compute active site residues involved in binding of product. We downloaded 1FHV in the last module. Download 1R6W and 1FHU for use in the present module. We will use these three structures for analysing the structure of free active site, structure of substrate bound active site and structure of product bound to active site.
Run SwissPdbViewer, and Open ‘1R6W’ PDB file. For detecting Enzyme Active Site residues, follow the procedure given in the last module “13 Analysis of Enzyme Active Site using SwissPDBViewer“. At the sake of repetition, follow these steps in order:
1. Select substrate ‘164735’ i.e. SHCHC, the last residue in the control panel, 2. From the ‘Tools’ menu, Select ‘compute H-bonds’ command
3. From the ‘display’ menu, select ‘Show Only H-bonds from Selection’ command 4. From the ‘display’ menu, select ‘Show Only groups with visible H-bonds’ command 5. From the ‘display’ menu, select ‘Render in Solid 3D’.
6. Press ‘Shift’ key and click in the third column of control panel to display labels. Uncheck label for substrate ‘164735’ and MG736, last two residues in control panel.
7. From the ‘display’ menu, select ‘Stereo View’ command 8. Click first button in the tool bar to center
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This will display 5 residues interacting with substrate through hydrogen bonding.
9. Display ASP161, LYS133 and LYS235 by clicking in the first column by marking ‘v’.
10. Display MG726, the second last residue, by clicking in first and fourth column in control panel.
11. Change the colour of last residue “164735” to cyan by clicking its box in sixth column of control panel
Enolate anion intermediate which is generated by two active site lysine residues located at position 133 and 235. The Mg2+ ion is octahedrally coordinated to ASP161, Glu190, ASP213, in active site and two water molecules with transition state stabilized by coordination of carboxylate group of the substrate to the essential Mg2+ ion in the sixth position.
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Go to Concept Map
3.3. Superposition or Structural Alignment of Enzymes
The structure of wild type enzyme without any ligand bound is available in 1FHU PDB fil. Therefore, this free form of the enzyme can be used to compare with OSBS structure with bound substrate and Mg2+ ion.
In case there is movement of active residues upon binding of the substrate, i.e. induced fit binding, then the active site residues in the structure with bound ligands will have different three dimensional positions than in the active site residue positions in the free enzyme. However, to compare three dimensional positions of the residues in two structures, we need to superimpose two structures. This is also known as structural alignment.
Now open the file 1FHU PDB file. This will open the structure of free enzyme. However, the complete structure is not visible. Therefore, click first toolbar button to center the molecules in display.
To achieve structure alignment, open ‘Fit’ menu, and select “Magic Fit” command.
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This will open “RMS & Auto Fit options" dialog box. Choose “Backbone Atoms only“ radioButton and select the free enzyme “pdb1fhu“ as fixed layer as well as “pdb1r6w“, the second layer.
Click OK button. This will superimpose two structures. Select “1FHU PDB“ file as active layer by opening the
‘Wind’ menu followed by selecting “Layers Info” command.
This will open the Layers Info window. Reposition this window to display complete window as shown:
Click on “pdb1fhu“ to select it as active layer to receive menu and toolbar commands.
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When more than one structure is opened in SwissPdbViewer, then each structure is loaded into a separate layer and each layer with loaded molecule is listed in “Layers Info” window. Hide “Layers Info” window. An active layer can also be selected from “Control Panel” window. Click in the bar displaying the name of the molecule of the current layer, i.e. “pdb1fhu“. This will open the prompt menu, with a check mark on
“pdb1fhu“ to convey that “pdb1fhu“ is the currently active layer.
To change the layer to “pdb1r6w“, just check it to make it active layer. The control panel will always display the sequence amino acid residues of currently selected layer, in the six columns of the control panel for its manipulation. At the moment, make “pdb1r6w“ as the current layer.
While “pdb1r6w“ file selected, open ‘Select’ menu and choose “None“ command.
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Now in the control panel, select residues 131, 133, 161, 163, 190, 213, 235 and 262. At the sake repetition select first residue i.e. 131 with a click on its number and then select rest of the residues with click while
“control“ key is also pressed. Now press Shift + left click in the first column to unmark each residue. Select and mark ‘v’ by clicking in the first column for residues 131, 133, 161, 163, 190, 213, 235 and 262. Follow the same procedure to select and display residues 131, 133, 161, 163, 190, 213, 235 and 262, while “1FHU PDB“ file selected.
Open display menu and uncheck three commands i.e. “Show Backbone as Carbon Alpha Trace”, “Show Backbone Oxygens” and “Show Sidechains Even When Backbone is hidden”, as shown next.
Now from control panel sixth column, press shift and click to set the color of 1R6W and 1FHU to green and red, respectively. Click the first button in toolbar menu to center the molecules.
The Stereovision of these active site residues in two enzymes, one free enzyme shown in red and other with bound substrate in green, shows that the red and green colours do not overlap. Consequently, there is some induced fit binding of the substrate ‘SHCHC’ to the OSBS enzyme.
Go to Concept Map
3.4. Quantifying Induced Fit Binding of ligands for enzymes having high specificity to substrate
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To quantify the extent of induced fit, one can calculate root mean squared deviation of three dimensional positions, i.e. RMSD between two structures. However, RMSD can be calculated when same groups are involved. Therefore, to calculate RMSD, select residues numbers 131, 161, 163, 190, 213, 235 and 262 by first selecting one layer. Repeat the same for second layer by selecting second layer and clicking on residue number 131 with subsequent clicks for residues numbers 161, 163, 190, 213, 235 and 262 with ctrl key pressed. Now select “Calculate RMS...” from Fit menu.
This will open the “RMS & Auto Fit options“ dialog box. Set the options as shown and click OK button.
This will display RMS in the status bar i.e. below tool bar, as 0.89 angstroms, which shows that there is induced fit binding of the substrate into the active site through movement of atoms in backbone.
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Now “Calculate RMS...”, for the shown parameters
This will display RMS in the status bar below tool bar as 1.54 angstroms, which shows that there is induced fit binding of the substrate into the active site through movement of each atom in side chains.
Therefore, there is more movement of side chain atoms than movement of backbone atoms.
Now “Calculate RMS...”, for the shown parameters.
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This will display RMS in the status bar below tool bar as 1.26 angstroms, which shows that there is induced fit binding of the substrate into the active site through movement of each atom in selected residues.
However, movement of 1.26 angstroms for each atom is in between 0.89 angstroms for movement of atoms in backbone and 1.54 angstroms for movement of atoms in side chains.
The residue at position number 133 in wild type is a lysine and an arginine in enzyme with bound substrate. Therefore select the Lys133 and Arg133 by clicking on these residues in each layer while “ctrl“
key pressed. Now calculate RMS with following parameter values for residues in two enzymes.
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This produces RMS of 0.93 angstroms for these residues, which is close to 0.89 angstroms for other residues selected. This shows that for backbone atoms movement is less than 1 angstroms, whereas, for side chain atoms the movement is more than 1 angstroms.
Now, select “1R6W PDB“ structure from layers info and click under ‘vis’ column to unmark ‘v’ to hide 1R6W structure.
Now close ‘Layers Info’ window.
Now let us compare free OSBS with OSBS bound to product OSB. From the file menu, select first Command “Open PDB File...” and select ‘1FHV’ PDB file containing OSBS bound to product OSB. However, to compare three dimensional positions of the residues in two structures, i.e. free OSBS with OSBS bound to product OSB, we need to superimpose two structures. To achieve structure alignment, open ‘Fit’ menu, and select “Magic Fit” command.
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This will superimpose two structures. Now select 1FHU and from control panel ensure that only eight residues are selected and visible, i.e. residue numbers 131, 133, 161, 163, 190, 213, 235 and 262. Now select 1FHV from control panel and ensure that only eight residues are selected and visible, i.e. same residue numbers 131, 133, 161, 163, 190, 213, 235 and 262. Press ‘Shift’ key and click in the third column of control panel to display labels. Click first button in the tool bar to center. This will display:
To calculate the RMSD between 131, 133, 161, 163, 190, 213, 235 and 262 residues with following parameters.
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The average RMSD between each atom is 1.47 angstroms.
Now “Calculate RMS...”, for the shown parameters.
The RMS between these residues is 1.77 angstroms.
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Now “Calculate RMS...”, for the shown parameters.
The RMS between these residues is 1.06 angstroms.
This proves again that there is more movement of side chain atoms than atoms in back bone, during induced fit binding.
From control panel select ‘1r6w’ and make it visible by checking ‘visible’ checkbox. Set color to ‘cyan’ using
‘shift press + click’ in the sixth column of control pane and selecting ‘Cyan’ color. Now ‘unmark’ residue number 133 in each of the three layers to display:
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Now, from control panel select ‘1fhu’ layer and make it invisible by unchecking ‘visible’ checkbox.
Now “Calculate RMS...”, for the shown parameters.
The RMS between these residues is 0.65 angstroms.
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This shows that the structures of the enzyme, i.e. one bound to substrate and other bound to product are near to each other and pass through a transition state bound active site which may be taken as an average of two i.e 0.325 angstroms away from each of one bound to substrate and other bound to product.
Now “Calculate RMS...”, for ASN163 between 1r6w and 1fhu
Now “Calculate RMS...”, for ASN163 between 1fhv and 1fhu
The OSBS is a highly specific enzyme binding only SHCHC and OSB. This shows that there is induced fit binding for the highly specific binding of ligands to enzyme active site.
Go to Concept Map
3.5. Induced Fit Binding in a broad specificity enzyme
Now let us take an example of Carboxypeptidase A, which is very broad specific enzyme. Carboxypeptidase A preferentially cleave C-terminal L-amino acids with aromatic or branched Sidechains. In addition, ester bonds of peptides with a free C-terminal carboxyl group can be cleaved. Further, Acylated -amino and -
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hydroxy carboxylic acids are also substrates. This will help us understand induced fit binding of ligands to broad specificity enzymes, which has to deal with varied structures of substrates and products.
Use “2CTC PDB“ file (http://www.rcsb.org/pdb/explore/explore.do?structureId=2CTC), which provides high resolution at 1.4 angstroms between Carboxypeptidase A and L-phenyl lactate. Use “1M4L PDB“ file (http://www.rcsb.org/pdb/explore/explore.do?structureId=1m4l) which provides high resolution at 1.25 angstroms for free enzyme structure. Open 2CTC and 1M4L with SwissPdbViewer and display the active site using Carboxypeptidase A bound to L-phenyl lactate in 2CTC and then superimpose free enzyme 1M4L using magic fit. Color L-phenyl lactate bound enzyme as green, free enzyme as red, L-phenyl lactate as Cyan and Zn2+ as grey, using Control panel sixth column boxes.
The literature shows that the conformational changes in the active centre of the enzyme upon binding of the inhibitor are restricted to only two residues, Tyr248 and Arg145. Mutation of Tyr248 to phenylalanine has no effect on the rate of catalytic reaction. Glu270 and coordinated Zn2+ ion are involved in breaking of bond. Display GLU270 and HIS69, GLU72, HIS196 coordinating Zn2+ ion, as these are involved in bond breaking. Display ARG145 involved in stabilizing interaction with C-terminal of substrate. Display Tyr248 involved in induced fit binding of the substrate, as shown above. Select these six residues using Control panel with first residue selected with a left click and subsequent residues selected with Ctrl + left click in both the structures. Do not select L-phenyl lactate and Zn2+ ion in 2CTC PDB file. This will have same six residues selected in both forms of the enzymes. This selection will be used for calculating RMSD.
From the Display menu, select “Calculate RMS” command with following parameters in the dialog box.
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This calculates RMSD for all atoms of the six selected residues.
This RMS of 2.79 angstroms for each atom in six residues is a very high value. Check the RMSD with all backbone atoms.
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This is just 0.24 angstroms.
This shows movement of specific residue side chains, which occurs with rotation around bond between C- alpha atom of main chain and first atom of side chain bonded to C-alpha atom of main chain. Check the RMSD of the residues HIS69, GLU72 and HIS196 involved in coordinating Zn2+ ion, which is involved in catalysis.
This is not different from overall RMSD. Now check, RMSD of the residues involved in binding ARG145 and TYR248 as well as the residue GLU270.
The three residue RMSD is very high. Now check the RMSD of ARG145 and GLU270:
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The RMSD of ARG145 and GLU270 is approximately one angstrom. Now check the RMSD of each of the three residues separately. For GLU270, it is 0.47 angstroms.
This shows that there is little movement of GLU270, which is involved in breaking of peptide bond.
Similarly ARG145 show movement approximately one angstrom.
However, TYR248 shows RMSD 6.24 angstroms
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Therefore, the main movement is for only Tyr248. To measure the distance i.e. length of movement of Tyr248 upon binding of the substrate, measure the distance between hydroxyl group of Tyr248 from both structures. To measure distance between two atoms, click fifth button in the toolbar.
Then first atom from which distance is to be measured is picked by clicking on the atom. This is followed by picking second atom by clicking on second atom.
There is movement of 12 angstroms by tyrosine upon binding. This is a big induced fit binding. Appreciate this induced fit change in StereoVision. But, first, using sixth column of control panel, with shift + left click, change the colours of free enzyme to red and substrate bound enzyme to green.
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Now measure the torsion angles of Tyr248 from both Free Carboxypeptidase A and Carboxypeptidase bound with Substrate. Select seventh tool in tool bar and pick one atom in the residue.
This shows that there is a specific movement of one residue involved in binding. However, this huge induced fit is through big rotation of torsion angle between bond formed by C-alpha and C-beta of Tyr248 side chain.
To rotate only bond between only C-alpha and C-beta of Tyr248 side chain from free enzyme, first select only Tyr248 from the free enzyme. To achieve same, first make 1M4L PDB file as active layer and select Tyr248 with a single left click.
Then make 2CTC PDB file as active layer using “Layers info” window or from Control panel “Top Bar”. Now select, command “None” from Select “Menu”.
In addition, change the movement from “Move All” to “Move Selection.
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This is achieved by clicking Move Toggle button.
We have seen that there is movement of tyrosine by 12 angstroms through specific rotation around carbon-carbon single bond between C-alpha and C-beta of methylene. One can rotate a bond through selecting last button for torsion tool, in the tool bar.
Now Pick C-beta of the methylene of tyrosine.
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This will display four pairs of arrows which can be clicked to change the torsion angles. First button will adjust phi, second will adjust psi. But Use third pair of button to adjust rotation around alpha and beta carbons. Fourth pair of button is used to adjust rotation around next bond i.e. beta and gamma carbons.
Therefore, there is specific rotation around C-alpha and C-beta carbon- carbon single bond of side chain to bind the substrate.
Go to Concept Map
3.6. Binding transition state structure to Active Site
We have seen the assumption of continuity in the transformation of enzyme active site structure, from free active site to substrate bound in active site to form ES complex. The ES complex then transforms substrate to transition state structure, bound to active site in ETS. Finally, this ETS complex transforms the transition state to product, bound to active site in EP complex and then regeneration of free enzyme with diffusion of product away from enzyme. This transformation of active site is used in the breaking and formation of bonds by an enzyme, during enzyme catalysed reaction. Till now we have seen only induced fit binding of ligands to enzyme active site. We have not seen the binding of transition state structure to active site. Ribonuclease A, an enzyme breaking phosphodiester bonds in RNA, complexes with Uridine Vanadate, a transition state analog. The pdb structure file ”1RUV”, contains high resolution (1.3 Angstrom) X-ray structure of Ribonuclease A, complexed with its transition state analog, Uridine Vanadate. Therefore, Download 1RUV PDB file and open using SwissPdbViewer.
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Four amino acids, GLN11, HIS12, THR45 and PHE120, are detected to form hydrogen bonds with 5 oxygens in Uridine Vanadate. These are GLN11, HIS12, THR45 and PHE120. The oxygen at ribose ring position 3 (labeled as first oxygen in cyclic transition state analog) do not form any hydrogen bond. Side chain of HIS12 acts as acid-base catalyst for the reaction. Side chain of HIS12 also stabilizes the oxygen at ribose ring position 2 (second oxygen in cyclic transition state analog) for binding to phosphorus to form a cyclic transition state. Third oxygen in the transition state analog is not bound to any side chain. Side chain of HIS119 acts as acid-base catalyst for the reaction by helping in breaking the phosphodiester bond to third oxygen backbone in the RNA substrate. Side chain of HIS12 also forms a hydrogen bond (1.86 Angstroms) with fourth oxygen in cyclic transition state analog bound. Main chain of PHE120 also forms a hydrogen bond (2.00 Angstroms) with fourth oxygen in the cyclic transition state analog. Side chain of GLN11 forms a hydrogen bond (2.07 Angstroms) with fifth oxygen in the cyclic transition state analog. Side chain of THR45 forms a hydrogen bond (2.11 Angstroms) with Uridine ring.
The transition state analog (Red), bound to active site of the enzyme in a cleft, is shown next.
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The total chemical synthesis of RNase A using modern chemical ligation methods was applied, illustrating chemical protein synthesis. The identity of the synthetic product was confirmed through rigorous characterization, including the determination of the X-ray crystal structure to 1.1 Angstrom resolution.
Download this PDB file 2E3W containing X-ray structure of native RNase A from Bos Taurus with 1.05 Å resolution and open using SwissPDBViwerer. First superimpose the two enzymes for all atoms in 124 amino acids selected in both structures through control panel and then Calculate RMS for all atoms while GLN11, HIS12, THR45 and PHE120 selected in both 2E3W and 1RUV.
This shows that free enzyme active site undergoes a change for each of the atoms in GLN11, HIS12, THR45 and PHE120 with average 0.31 Å movement to reach transition state. Now select all 124 amino acids in each of both 2E3W and 1RUV and then Calculate RMS for all atoms
This shows that free enzyme active site undergoes a change for each of the atoms with average 0.98 Å movement to reach transition state. Therefore, there is more change in other atoms. Now select HIS12 and HIS119 amino acids in each of both 2E3W and 1RUV and then Calculate RMS for all atoms
This shows that free enzyme active site undergoes more change for each of the atoms in HIS12 and HIS119 amino acids with average 2.11 Å movement to achieve transition state. Therefore, there is more movement of HIS12 and HIS119 amino acids i.e. amino acids involved in actual catalysis.
Therefore, In the bidirectional i.e. reversible reaction, four structures of the same enzyme are involved:
these include
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1. Enzyme bound to solvent, i.e. free enzyme in the solvent, 2. Enzyme bound to substrate,
3. Enzyme bound to transition state structure and 4. Enzyme bound to product.
This bidirectional reaction cycle is able to achieve equilibrium of each enzyme form, in reversible reactions.
The structures are represented in the cartoon form, shown next
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Each of the four structures of enzyme has geometric and electronic Complementarity to its bound ligand(s).
Go to Concept Map 4. Summary
Dear students, we know that there are three models for understanding enzyme action. These are lock &
Key, Induced fit and transition state model. In this module, we learnt that enzyme catalysed Reaction are reversible i.e. bidirectional. In each direction of the reversible reaction i.e. forward and reverse direction, there are four steps each: For the forward direction, the first step involves diffusion of the substrate to the active site of the free enzyme for formation of enzyme -substrate (ES) complex. The substrate bound enzyme complex then achieves a transition state structure bound to enzyme (ETS). This ETS complex then yields an enzyme-product (EP) complex. The product then finally diffuses away from the enzyme and the free enzyme conformation is regenerated. To achieve catalysis, four structures of the same enzyme, bound to its different ligands are involved. Each of the four structures of enzyme active site has geometric and electronic Complementarity to its bound ligands i.e. solvent in free enzyme, substrate in ES complex, transition state in ETS complex and product in EP complex. We have learned to compare three dimensional positions of the residues in two structures through superimposing two structures, i.e. structural alignment.
The structural alignment revealed that there is some induced fit binding for specific binding of highly ligands to enzyme active site. On the other hand, there is very high specific movement of side chain s for binding the substrate to a broadly specific enzyme, such as Carboxypeptidase A. Further, free enzyme active site undergoes more change for each of the atoms, actually involved in chemical catalysis, to reach transition state.
