Question | Answer |
enzyme | - the name will usually end in -ase (ex. catalase, alpha-amylase, tyrosinase)
- protein that typically catalyzes (facilitates) reactions
- not altered or consumed by the reaction |
Why do reactions need to be ‘facilitated’? | - most biological chemical reactions only occur at meaningful rates when in the presence of an enzyme because of the activation energy barrier
- basically, a certain amount of E is required to start a chemical reaction; enzymes lower that |
What changes in the presence of the enzyme? | the activation energy (E.A) |
active site | the region of an enzyme where substrate molecules bind and undergo a chemical reaction |
substrate | - the material upon which an enzyme acts
- what gets converted into products |
Enzymes bind and stress reactants to catalyze reactions through ___, ___, and ___. | - orientation
- physical strain
- chemical change |
What affects rates of enzyme controlled reactions? | - the physical and chemical environment of the enzyme (pH, temperature, etc.)
- availability of necessary co-factor
- presence of molecules that act as inhibitors
- relative concentration of substrate, enzyme, and product |
Because pH also influences shape and therefore reaction rate, each enzyme has a(n) ___. | optimal pH |
How does temperature affect enzyme reaction rate? | - as temp. increases, molecules move faster, so collisions between substrates and active sites occur more often
- however, at some point, thermal agitation begins to disrupt the weak bonds holding the protein together and the protein eventually denatures |
Some enzymes require ___ to function normally. These are either metal ions or small organic molecules called ___. | - cofactors
- coenzymes |
How does substrate concentration (SC) affect enzyme reaction rate? | - the rate of product formation increases linearly at low SC
- the reaction rate reaches maximum speed at high SC (called V.max)
- at V.max, all active sites on the enzymes are occupied, so no more product can be made |
What is tyrosinase? What are some examples of its effect? | - an enzyme that catalyzes the biosynthesis of the pigment melanin from the amino acid tyrosine
- skin coloration, browning of fruits |
What is the cofactor of tyrosinase? | copper ions |
Explain how dopachrome is produced. | - in the presence of O2, tyrosinase (E) catalyzes the hydroxylation of tyrosine into DOPA
- E then catalyzes DOPA into dopaquinone
- dopaquinone then spontaneously converts into dopachrome |
You can monitor the activity of tyrosinase by looking at the color of the solution. Over time, what do you think will happen to the color of a DOPA solution after the addition of tyrosinase? | - the solution will begin clear and then turn brown
- will get darker the longer the reaction proceeds |
spectrophotometer | an instrument which measures the amount of light (of a specificed wavelength) which passes through a medium |
How does a spectrophotometer work? | - a cuvette containing the solution is inserted
- light passes thru the cuvette/solution and then strikes a detector
- detector records what light is transmitted
- using this info, the wavelengths that were absorbed can be determined |
Once you find a product's peak absorbance (or maximum absorbance wavelength) what do you use it for? | to measure enzyme activity |
What does tyrosinase convert more of: DOPA or tyrosine? | tyrosinase is much better at converting DOPA to dopaquinone than it is at converting tyrosine to DOPA |
In exercise 4, where did you find the enzyme? The substrate? | - the enzyme tyrosinase was found in a potato
- the substrate (DOPA) was in a stock solution |
serial dilution | - prepared when the set of dilutions range over at least two orders of magnitude
- geometric progression
- ex: 20 mM -> 10 -> 5 -> 2.5 -> 1.25 mM |
denaturation of an enzyme | - the loss of catalytic activity when placed in a hostile environment
- enzyme remains physically intact (just modified) |
What is a blank and what is it used for? | - contains all of the components of the solution except the test substance
- to calibrate a spectrophotometer |
In section 4.2, you used a blank containing a buffer solution and potato homogenate (but no DOPA) to calibrate the spectrophotometer. Why is this blank necessary? | - to be able to detect JUST the effect of E on ab. (not reactants or buffer), the spec. must be zeroed using a solution w/ everything but DOPA
- when the cuvette w/ E is placed in the spec. any change in ab. that is detected is due solely to the product |
Your hypothesis is: "Lowering the pH of solution containing tyrosinase will decrease the rate at which it catalyzes the production of dopachrome from DOPA." What is the null hypothesis? Negative/positive controls? Independent/dependent variables? | - NH: "Lowering the pH will not affect the enzymatic activity of tyrosinase."
- NC: no enzyme run (buffer + DOPA)
- PC: normal run (buffer + DOPA + tyrosinase)
- IV: pH of solutions
- DV: rate of reaction / dopachrome production |
In lab you used the SpectroVis Plus Spectrophotometer to measure the light that was absorbed by dopachrome, a dark orange/brown molecule. Is a solution that appears dark orange in color absorbing orange wavelengths of light? | - no, it is REFLECTING orange wavelengths of light
- (it is absorbing blue wavelengths of light; ~470 nm) |