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what are some characteristics special to enzymes?
Enzymes are biological catalysts that speed up chemical reactions without being consumed in the process. They are highly specific, operate under optimal conditions, and lower the activation energy required for reactions.
what is the substrate?
the molecule that the enzyme converts into products
how do enzymes speed up reactions?
by lowering the activation energy needed for the reaction to occur. they position the substrates, adjusting their bonds so that they become unstable and reactive to form products more easily.
what is the activation energy?
the amount of energy required to initiate a chemical reaction
Explain why an enzyme can increase the reaction rate compared to an uncatalyzed reaction.
Enzymes increase the reaction rate by lowering the activation energy, allowing more substrate molecules to reach the transition state and form products in a shorter time compared to uncatalyzed reactions.
what are 3 factors that can affect the rate at which an enzymatic reaction occurs?
temperature
pH
substrate or enzyme concentration
generally what do lower temperatures do to enzymatic reactions?
slow them down
generally what do higher temperatures do to enzymatic reactions?
speed them up because it increases the kinetic energy for more molecule interactions
what is a cofactor
A non-protein chemical compound that is required for the biological activity of a protein, often an enzyme. Cofactors can be metal ions or organic molecules known as coenzymes.
what is cellobiase?
An enzyme that hydrolyzes cellobiose into glucose molecules.
what is the natural substrate for the enzyme cellobiase?
cellobiose (disaccharide composed of 2 beta glucose molecules)
why is p-nitrophenyl glucopyranoside used instead of cellobiose?
there are not many simple, inexpensive, fast methods to detect the molecule quantitatively
what happens when the artificial substrate is broken down by cellobiase?
it produces glucose and p-nitrophenol as products of the reaction.
what happens when p-nitrophenol is mixed with a solution that is basic in pH? (stop solution)
it turns yellow
what is the amount of yellow color produced proportional to?
the amount of p-nitrophenol present
what does the deeper yellow color indicate?
a higher amount of product made
what is one simple method of estimating how much product is formed is?
by comparing the yellowness of enzyme reaction samples to a set of known standards, which contain a known amount of colored product.
what does a spectrophotometer do?
quantitatively measures the amount of yellow color by shining a beam of light (wavelength of 410 nm) through the sample. The spectrophotometer measures the amount of light that is absorbed by the sample
what happens in the spectrophotometer the darker the color of yellow the sample is?
the more light that is absorbed and thus the more concentrated the sample
what is determined first in order to determine what factors influence an enzyme's ability to break down its substrate?
the rate of reaction or how much product is formed in a set amount of time
in the lab to study cellobiase activity, what did we measure?
the rate of reaction by adding enzyme to the artificial substrate p-nitrophenyl glucopyranoside
in the lab, what were the enzyme and substrate dissolved in?
a buffer that is at an ideal pH (pH 5.0) for the reaction to occur
how can the reaction rate be calculated?
by calculating how much p-nitrophenol is produced over time
Briefly explain how a spectrophotometer works. Or alternatively describe what it measured during the enzymatic reactions we did in class.
A spectrophotometer measures the intensity of light absorbed by a sample at specific wavelengths. During the enzymatic reactions, it quantified the concentration of p-nitrophenol produced.
In the enzyme lab, we plotted known enzyme concentrations as X-coordinates and their respective absorbances as Y-coordinates. For the standard curve, this resulted in what kind of line and what does that line tell you about the relationship between concentration and absorbance?
A linear line, indicating a direct proportionality between enzyme concentration and absorbance.
In the enzyme lab experiment, we had a control tube that did not contain the enzyme cellobiase. But the tube still had the substrate (p-nitrophenyl glucopyranoside) and buffer. What should you have seen when you measured the absorbance values at the start (0 min) and end (8 min) of the reaction.
You should have seen no significant change in absorbance values, indicating that without the enzyme, there was little to no conversion of the substrate to p-nitrophenol.
We observed that more product could be produced over time as our enzyme cellobiase converted p-nitrophenol glucopyranoside into glucose and p-nitrophenol. If we kept on taking absorbance measurements after the 8 min mark, what would you expect to see and why.
You would expect to see a plateau in absorbance values after a certain point, indicating that the enzyme has reached its maximum activity and the substrate is becoming limiting.
Did you observe any changes in the enzyme reaction and control reaction conical tubes during the time that the reaction was occurring?
Yes, in the enzyme reaction tube, the solution gradually changed from clear to yellow over time as p-nitrophenol was produced. In contrast, the control reaction tube (which contained no enzyme) showed little to no color change, indicating that the reaction did not proceed significantly without the enzyme.
What happened to the solution in each cuvette after you added the enzyme/substrate mixture to the stop solution?
The stop solution (a strong base) caused the p-nitrophenol in the cuvettes to turn yellow. The intensity of the yellow color correlated with the amount of p-nitrophenol present, allowing for a quantitative measurement using a spectrophotometer.
Describe the chemical reaction that occurred in this experiment.
The enzyme cellobiase catalyzed the hydrolysis of p-nitrophenyl glucopyranoside into glucose and p-nitrophenol. The p-nitrophenol then reacted with the basic stop solution, turning yellow, which enabled absorbance measurements at 410 nm
Describe the amount of product produced in the enzyme-catalyzed reaction compared to the control where no enzyme was added.
The enzyme-catalyzed reaction produced a significant amount of p-nitrophenol, as shown by increasing absorbance values over time. In contrast, the control reaction (without enzyme) showed little to no absorbance increase, indicating that no significant product was formed
If you took a time point at 15 minutes, do you think more product would be produced than at 8 minutes? Explain your answer.
It is unlikely that a significant amount of additional product would be produced at 15 minutes compared to 8 minutes. Over time, substrate depletion, enzyme saturation, or product inhibition can slow down the reaction. If all available substrate has already been converted, the reaction would have reached a plateau.
how did you estimate the amount of product produced by the enzyme?
The amount of p-nitrophenol was determined using a standard curve, which correlates absorbance values at 410 nm with known concentrations of p-nitrophenol. By comparing the reaction sample absorbance values to this curve, the nmol of product formed was calculated
Why is the amount of light absorbed by the sample proportional to the amount of product produced?
p-Nitrophenol absorbs light at 410 nm. As more p-nitrophenol is produced, the yellow color intensifies, leading to higher absorbance values. This follows Beerās Law, which states that absorbance is directly proportional to the concentration of the absorbing species.
Is the rate of product production constant over time? Hint: Is the slope of the line constant or does it change?
No, the rate is not constant over time. The slope of the p-nitrophenol production vs. time graph changes, indicating that product formation slows down as the substrate is consumed, the enzyme becomes saturated, or product inhibition occurs.
what is Michaelis Curve?
A graph that illustrates the relationship between the reaction rate of an enzyme and the substrate concentration, typically showing a hyperbolic shape. It demonstrates how enzyme activity increases with substrate concentration until it reaches a maximum velocity (Vmax) when the enzyme becomes saturated.
what is Vmax?
The maximum rate of reaction achieved by an enzyme when it is saturated with substrate, reflecting the enzyme's catalytic efficiency.
what is Km?
The Michaelis constant, Km, is the substrate concentration at which the reaction rate is half of Vmax. It provides insight into the affinity between an enzyme and its substrate, with lower Km values indicating higher affinity.
How would the addition of enzyme/substrate/product affect these values (Michaelis Constant, Vmax, and Km)? What about inhibitors?
Enzyme: Increases Vmax (more active sites). Km is same (unchanged affinity)
Substrate: no effect on Vmax or Km
Product: if it does not inhibit, no direct effect on either
INHIBITION:
Competitive: decreases Km and Vmax is unchanged
Noncompetitive: Km is unchanged and V max decreases
Uncompetitive: both decrease
Note 
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