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Enzyme with long tube leading to active site?

Enzyme with long tube leading to active site?


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Are there any enzymes that force one or more of their substrates to travel a relatively long distance through a small opening/tube through the enzyme and to the active site? (not a transport protein)


First of all, as you mentioned, there are a lot of membrane / transport proteins with very nice, well defined tunnels. But you're looking for a soluble / cytosolic enzyme.

Some oxygen-dependent enzymes have quite long tunnels that oxygen is using. For example this one: http://www.jbc.org/content/283/36/24738.short or this one: http://europepmc.org/articles/PMC2698890

Then there are some enzyme with just deep active sites, I think the p450 BM3 is quite known for that, but any enzyme with a reasonable specificity (and especially those that have very reactive cofactors) will have a "tunnel".

You can also find long "tunnels" in enzymes that accept long substrates. I like this example of squalene synthase: http://science.sciencemag.org/content/277/5333/1811.full. The substrate first has to go deep into the enzyme to find the proton it needs to start a quite interesting reaction.

If you want to find new tunnels in your enzyme there are also tools available, such as CAVER: http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002708 If you look at the papers citing this tool you might also find some cavities.


Living organisms sustain the activities of life by carrying out thousands of chemical reactions each minute. These reactions do not occur randomly, but are controlled by biological catalysts called enzymes. Enzymes accelerate the rate of chemical reactions by lowering the activation energy needed to trigger the reaction. Without enzymes, chemical reactions would not occur fast enough to support life. Enzymes are typically proteins and each is composed of a specific sequence of amino acids. Hydrogen bonds form between specific amino acids and help create the 3-dimensional shape that is unique to each enzyme. The shape of an enzyme, particularly its active site, dictates catalytic specificity of a particular enzyme. Each enzyme will only bind with specific molecules, as these molecules must fit with the active site on the enzyme like a lock and key.

A molecule that binds with an enzyme and undergoes chemical rearrangement is called a substrate. The enzyme &ldquoE&rdquo combines with the substrate molecule(s) &ldquoS&rdquo at the active site and forms a temporary enzyme-substrate complex &ldquoES&rdquo, where the specific reaction occurs. The modified substrate molecule is the product &ldquoP&rdquo of the reaction. The product separates from the enzyme and is then used by the cell or body. The enzyme is neither consumed nor altered by the reaction and can be used in other catalytic reactions as long as additional substrate molecules are available.

An individual enzyme molecule may facilitate several thousand catalytic reactions per second, and therefore only a small amount of enzyme is needed to transform large amounts of substrate molecules into product. The amount of a particular enzyme found in a cell at any given time is relative to the rate at which the enzyme is being synthesized compared to the rate at which it is degraded. If no enzyme is present, the chemical reaction catalyzed by that enzyme will not occur at a functional rate. However, when the concentration of the enzyme increases, the rate of the catalytic reaction will increase as long as the substrate molecules are accessible.

Various factors can inactivate or denature enzymes by altering their 3-dimensional shape and inhibiting their substrate binding efficiency. Many enzymes function best within a narrow temperature and pH range as substantial changes in temperature or pH disrupt their hydrogen bonds and alter their shape. Change in enzyme shape typically alters the shape of the active site, and affects its ability to bind with substrate molecules. It is the unique structural bonding pattern of an enzyme that determines its sensitivity to change in temperature and pH.

In the following exercise you will explore the effects of pH and temperature on the activity of the enzyme amylase. Amylase is found in the saliva of humans and other animals that consume starch as part of their diet. Starch is a plant polysaccharide composed of many glucose molecules bonded together. Amylase controls the initial digestion of starch by breaking it down into disaccharide maltose molecules. Maltose is ultimately broken down into glucose molecules in the small intestine when other enzymes are utilized.

Figure 1: Branching structure of starch.

The rate at which starch is digested into maltose is a quantitative measurement of the enzymatic reaction. The rate of starch degradation is relative to the rate at which maltose is produced, however it is easier to test for the presence of starch than it is to measure the rate of maltose production. I2KI will be used as indicator for the presence of starch. When starch is present, I2KI turns a blue-black color. In the presence of maltose, I2KI will not react and remains an amber color.

[ Starch + I_2KI ightarrow ext onumber]

[ Maltose + I_2KI ightarrow ext onumber]

Your group will be assigned to conduct one or more of the following activities (in part or whole):

I. The effect of pH on amylase activity

II. The effect of temperature on amylase activity

Results from each exercise will be presented to the class and students will be responsible for the information and results from all exercises.


Introduction

Efficient de novo computational enzyme design has been a long-held goal of protein engineers and would allow the catalytic power of enzymes to be directed towards a range of industrially and medically important chemical reactions. Studies have demonstrated that although de novo design is possible, the imperfect designs often require optimization through laboratory evolution 1,2 . Our ability to design enzymes rests upon our fundamental understanding of enzyme catalysis, yet the biophysical and chemical basis for their catalytic efficiency remains a topic of debate 3,4,5 . There is evidence for contributions to catalysis from electrostatic transition state (TS) stabilization, conformational changes, and quantum tunneling 6,7,8 . Conformational sampling has been shown to allow enzymes to adopt specific configurations that are suited to different steps in their catalytic cycle and recent work has shown how remote mutations can alter the conformational landscape to increase sampling of certain conformational substates 7,9 . Vibrational motions have also been suggested to contribute to the chemical step in catalysis by altering the probability of transmission through the TS barrier in some enzymes by quantum mechanical hydrogen tunneling 8,10 .

Kemp elimination (proton elimination from 5-nitrobenzisoxazole Fig. 1) has been extensively used as a model system in enzyme design owing to the simplicity of the base-catalyzed ring opening reaction 11 and the absence of natural Kemp eliminases 1 , although some enzymes have been shown to catalyze Kemp elimination promiscuously 12,13 . Computational design of KE07 involved construction of a theozyme to catalyze the chemical reaction, which was then grafted into the scaffold of imidazole glycerol phosphate synthase (HisF) from Thermotoga maritima 1 . Catalytically essential residues from the initial design include a base (Glu101) that facilitates C−H bond cleavage, an H-bond donor (Lys222) to stabilize the phenoxide intermediate, and a π-stacking residue (Trp50), which was designed to stabilize the transition state and favor substrate binding through interactions with the aromatic ring of the substrate. This initial KE07 design (Round 1 R1) catalyzes the cleavage of 5-nitrobenzisoxazole (1), with 10 3 -fold rate acceleration over the noncatalyzed reaction and a turnover rate (kcat) of 0.018 s −1 . Seven generations of directed evolution then enhanced this turnover-rate over 100-fold 1 .

Reaction scheme for the Kemp elimination of 5-nitrobenzisoxazole. The nucleophilic oxygen atom of the base (B) donates electrons to the electrophilic 3′-H of 5-nitrobenzisoxazole and the electronegative oxygen atom of the isoxazole group forms a hydrogen bond with an acid (A) (1), forming a transition state in which the C−H and N−O bonds are weakened (2). The removal of the 3′-H from the substrate leads to an anionic phenoxide intermediate, which is then protonated, forming the final product (3)

Although the improvements to KE07 have been partially rationalized through experimental and computational characterization of the mutant proteins 14,15,16,17 , accounting for the effects of remote mutations in later rounds has been challenging. KE07 is not the most efficient of the several Kemp eliminases now designed 18,19,20 , but in the context of understanding how enzyme activity can be gradually improved through stepwise mutations, its low efficiency makes it an ideal model system to study the mechanisms by which evolution or engineering can improve an inefficient starting point.

In this study we use a combination of protein crystallography, enzyme kinetics, and computational approaches to investigate the structure, function, and dynamics of a series of improved variants of the KE07 series. By soaking crystals of various KE07 variants with substrate, we capture the enzymes with a series of different active site configurations. Using molecular dynamics simulations to investigate the sampling of the different conformational substates, we show that the evolutionary improvement of KE07 involves conformational selection of an alternative, nondesigned, active site configuration.


Watch the video: Enzyme Aktualisiert (February 2023).