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<!doctype html>
<html lang="en">
<head>
<meta charset="utf-8">
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<title>Presentation</title>
<meta name="description" content="BI281H Discussion">
<meta name="author" content="Michael J. Harms">
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<div class="slides">
<section>
<h2>Michaelis-Menten kinetics</h4>
<h4>2017-10-20</h4>
<br/>
</section>
<section>
<p>What do enzymes do to the reaction coordianate to speed up reactions?</p>
<p class="fragment" style="color:blue">They lower the activation energy by stabilizing the transition state</p>
</section>
<section>
<p>_____________ changes in activation energy lead to ____________ changes in rate.</p>
<ol>
<li>Small, large</li>
<li>Small, Small</li>
<li>Large, Small</li>
<li>Large, Large</li>
</ol>
<p class="fragment" style="color:blue">1 and 4</p>
<p class="fragment" style="color:blue">Stabilizing TS by $67 \ kJ \cdot mol^{-1}$ gives a 100-billion fold increase in rate!</p>
</section>
<section>
<p>What part of the serine protease enzymatic mechanism does the most to speed up the reaction?</p>
<p class="fragment" style="color:blue">Oxyanion hole: 2 hydrogen bonds to the negatively charged intermediate</p>
</section>
<section>
<p>How do organisms regulate the rates at which they do chemistry?</p>
<a href="presentation-data/01/img/roche-metabolic_pathways.png"><img src="presentation-data/01/img/roche-metabolic_pathways_small.png" /></a>
<small color="gray" align="left">Gerhard Michal, Roche</small>
</section>
<section>
<img src="presentation-data/12/img/proteins.jpg" height="80%" width="80%"/>
</section>
<section>
<h4>Conceptual goals</h4>
<ul>
<li class="fragment">Understand the properties of Michaelis-Menten enzymes</li>
<li class="fragment">Understand how experimental rate measurements allow determination of MM parameters</li>
<li class="fragment">Understand how cells tweak these parameters to achieve regulated activity</li>
</ul>
<h4>Skill goals</h4>
<ul>
<li class="fragment">Interpret experimental graphs for MM enzymes in terms of altered MM parameters.</li>
<li class="fragment">Interpret changes to those parameters in terms of altered underlying chemistry.</li>
</ul>
</section>
<section>
<p>Resources</p>
<p>Homework (do it...)</p>
<p>Lab 5</p>
<p>Practice problems later</p>
<p>Simulator <a href="http://aclarke.uoregon.edu:8000">http://aclarke.uoregon.edu:8000</a></p>
</section>
<section>
<p>$E \cdot S \color{red}{\rightarrow} E + P$</p>
<div class="fragment">
<p>Expand the arrow:</p>
<p>$E \cdot S \color{red}{\rightleftarrows E \cdot TS \rightarrow } E + P$</p>
</div>
<p class="fragment">$E \cdot S \color{red}{\overset{k_{cat}}{\rightarrow}} E + P$</p>
<p class="fragment">$k_{cat}$ is the rate constant for the reaction once substrate is bound</p>
</section>
<section>
<p>$velocity = V = [E \cdot S] k_{cat}$</p>
<p class="fragment">What determines $[E \cdot S]$?</p>
<ul class="fragment" style="color:blue">
<li>$[E]_{T}$: the total enzyme concentration</li>
<li>$[S]$: the substrate concentration</li>
<li>$K_{M}$: the "affinity" of the enzyme for substrate</li>
</ul>
<p class="fragment">$V = [E \cdot S] k_{cat} = [E]_{T} \theta_{ES} k_{cat}$
</section>
<section>
<p>What is $\theta_{ES}$?</p>
<div class="fragment">
<p>$E \cdot S \overset{K_{M}}{\rightleftarrows} [E] + [S] $</p>
<p>$\theta_{ES} = \frac{[ES]}{[E] + [ES]}$</p>
<p>$\theta_{ES} = \frac{1}{1 + K_{M}/[S]}$</p>
</div>
<p class="fragment">$K_{M}$ (the Michaelis constant) is basically a $K_{D}$. It measures the affinity of the enzyme for substrate.</p>
</section>
<section>
<p>The Michaelis-Menten equation:</p>
<p>$V_{0} = k_{cat}[E]_{T}\frac{1}{1 + K_{M}/[S]_{0}}$</p>
<p class="fragment">The velocity of a reaction is determined by:</p>
<ul class="fragment">
<li>The rate constant for the enzyme when bound to substrate ($k_{cat}$)</li>
<li>And the concentration of substrate bound to enzyme. This is determined by $K_{M}$, $[S]_{0}$, and $[E]_{T}$.</li>
</ul>
</section>
<section>
<p>$E + S \color{blue}{\overset{K_{M}}{\rightleftarrows}} E \cdot S \color{red}{\overset{k_{cat}}{\rightarrow}} E + P$</p>
<p>Would a mutation to serine protease disrupting the oxanion hole alter $k_{cat}$ or $K_{M}$?</p>
<p style="color:red" class="fragment">$k_{cat}$ would go down. It would disrupt ability to hop over transition state.</p>
<p class="fragment">How would it alter the $V$ vs. $S$ curve?</p>
<p style="color:red" class="fragment">It would lower the maximum rate, but leave shape unchanged.</p>
</section>
<section>
<p>$E + S \color{blue}{\overset{K_{M}}{\rightleftarrows}} E \cdot S \color{red}{\overset{k_{cat}}{\rightarrow}} E + P$</p>
<p>Would a mutation to serine protease disrupting binding of the peptide alter $k_{cat}$ or $K_{M}$?</p>
<p style="color:blue" class="fragment">$K_{M}$ would go up. It would disrupt ability to bind substrate.</p>
<p class="fragment">How would it alter the $V$ vs. $S$ curve?</p>
<p style="color:blue" class="fragment">It would raise the $K_{M}$, but leave the maximum rate unchanged.</p>
</section>
<section>
<p>Summary I</p>
<ul>
<li class="fragment">Enzyme chemistry can be described by:<br/>$E + S \rightleftarrows ES \rightarrow E + P$</li>
<li class="fragment">Initial enzyme velocity ($V_{0}$) is:<br/>$V_{0} = k_{cat} \times [E]_{T} \times \theta$</li>
<li class="fragment">$k_{cat}$ is the "intrinisic enzyme rate" in $s^{-1}$</li>
<li class="fragment">$[E]_{T}$ is the total enzyme concentration</li>
<li class="fragment">$\theta$ is the fractional saturation of the enzyme with substrate</li>
<li class="fragment">$\theta = \frac{[S]_{0}}{K_{M}+[S]_{0}} = \frac{1}{1 + K_{M}/[S]_{0}}$</li>
<li class="fragment">$K_{M} \approx K_{D}$ for substrate</li>
</ul>
</section>
<section>
<p>Summary II</p>
<ul>
<li class="fragment">Michaelis-Menten equation is:<br/>
$V_{0} = k_{cat} [E]_{T} \frac{[S]_{0}}{K_{M} + [S]_{0}} $</li>
<li class="fragment">Mucking up enzyme chemistry lowers $k_{cat}$</li>
<li class="fragment">Mucking up substrate binding increases $K_{M}$</li>
</ul>
</section>
</div>
</div>
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