Lifecycle of an Enzyme: Denatured

Lifecycle of an Enzyme: Denatured

If the tale of a brewing enzyme begins with hydration and synthesis in the aleurone layer of the barley kernel, where exactly does it end? The simple answer is, in my belly...but that’s not exactly the whole story. 

Most enzymes are simply amino acids bound together as proteins. With a very specific shape, they are able to catalyze certain chemical reactions, sometimes incredibly fast. The types of amino acids and the ways in which they link add specificity to the shape. Other chemical bonding, like those from metal ions, can influence the shape, and more importantly the “active site” of the enzyme.

Enzymes are relatively large in comparison to the substrates on which they work. Often a very small area on the enzyme encompasses the active site, which is where the substrate will temporarily bind when splitting or binding the catalyzed molecules. The entirety of an enzyme can encompass anywhere from 62 to over 2,500 amino acid residues, but only 2-4 tend to be directly involved in the active area. 

One important thing to understand about enzymes is that they are not “used up”. An enzyme will continue to perform its primary function as long as substrate is available, and as long as it is able to hold the specific 3D shape. No number of catalyzed reactions will weaken or destroy the enzyme itself. To that same end, enzymes do not change the equilibrium of a substrate. The chemical components of the system will remain in the same quantity, although the orientation may change. Enzymes do not add or subtract from the overall content. Further resources are available on this site, but consider clicking over to the MBAA for more information.

Inhibitors do exactly what they sound like and inhibit the activity of an enzyme. Whether competitive or non-competitive, they change the orientation of the enzyme shape as to make it unavailable for binding or splitting molecules. Competitive inhibitors are substances which “compete” for the active site of the enzyme. In the most basic sense, they fill the shape on the active site and block other substrate from entering. Enzymes do not “lock up” with the substrate or inhibitor, so to overcome a competitive inhibitor, more substrate is the answer. Increasing the substrate will provide a higher ratio as compared to the inhibitor, and therefore more beneficial reactions will take place.

Non-competitive inhibitors are a bit different. Rather than binding to the active site, these compounds link to other points on the enzyme structure. This causes a shift in the 3D shape of the enzyme, and can eliminate the ability for intended reactions. More substrate will not overcome the inhibition, as the shape is essential to facilitate a reaction. Substrate is not able to bind in the active site, and therefore no reaction takes place.

There are uncompetitive inhibitors and mixed inhibitors as well, however this leads us a bit further down the rabbit hole. For simplicity's sake, we will say it is also possible for an inhibitor and enzyme to link during the process of catalyzation, prohibiting the end result. If molecules binding on an enzyme do not affect the enzyme catalyzation, then they are not considered an inhibitor.

Irreversible inhibition is often referred to as denaturation. Covalent bonds or other strong bonds permanently bind the inhibitor to the active site of the enzyme, stopping all possibility for catalytic reactions. Think a competitive inhibitor that is really, really tough. It just does not let go. 

Activators are on the other end of the spectrum. Substances which increase the rate of reactions an enzyme undergoes. Think of these as “boosters” that help the enzyme work more efficiently. Glutamine is one activator that increases reactions in salivary amylase, breaking down starch faster. This results in foods with high starch content, like corn, tasting sweet on the tongue. Sometimes substrates themselves act as activators. When an active site is partially filled by one substrate, another may become more likely to fill in the active site and complete a reaction. This is due to the polarity and orientation of the combined enzyme and substrate drawing a stronger attraction.

Temperature is the primary cause of concern when it comes to denaturation. Enzymes tend to see a modest exponential growth of activity as they approach optimal temperature, however once above the optimal temperature, they see a sharp exponential decline. This quickly results in the enzyme being ineffective. For this reason, it is always best to slowly approach an optimum temperature. Going over by even a little can have serious consequences. Endogenous (from within barley) enzymes are quite sensitive to temperature, but exogenous (outsourced) options have been selected to withstand more fluctuation.

Second to temperature, pH of an enzymatic solution is essential. Litmus test strips are okay, but a MW102 from Milwaukee Instruments is a better option. The hydrogen ion concentration (pH) of a solution has a large impact on molecular chemical bonding. Basically, magnetic forces push or pull the structure of an enzyme into unusual shapes, changing the fit of the active site. Ions in the mash can help rigidize the structure of an enzyme, buffering it against some pH change, and preserving the active site. In most all-barley mashes necessary components are provided by the malt, however it may be beneficial to add some ions for different purposes.

If an enzyme can be denatured by changing its shape, then is it possible to renature it? Logical thought would indicate that a simple “reforming” of the 3D structure would render an enzyme effective again. This would be correct. The problem lies when an enzyme has been denatured so badly that it cannot “reform”. Coagulation of proteins occurs when an extreme level of denaturation occurs. Basically, the changed structure of the protein matrix causes binding with other protein formations, and the “sticking together” renders them unable to be pulled apart without destroying the original amino chains. 

Need a visual example? - Look into a boiling wort kettle. “Flocks” of hot break represent proteins that have stuck together due to denaturation. Are these flocks small, sandy-like grains? Or perhaps long, stringy ropes? Measure the pH. You might be surprised to see the role pH plays in the formation of the hot break. Coagulation is irreversible, so be careful with high heat and extreme pH levels!

 

Meet the Author

J.D. Angell

Meet the Author

J.D. Angell

After several years of providing hazardous materials training and maintenance for the world's largest brewing facility, JD began home brewing countless varieties of craft beer. Some early success and a detour with industrial scientific research engaged his interests in industrial equipment and complex science, while working at a liquid yeast supplier pointed him specifically towards enzymes. Currently heading Bircus Brewing Company in Ludlow, KY, JD blends contemporary flavors with traditional science and innovative techniques. With over a decade of operational brewing and independent contracting experience across 5 time zones, he has amassed a plethora of knowledge to share with fellow brewers. 

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