CHEM 146 Journal Article Study Sheet

Article 2: Binstock JF, Pramanik A and Schulz H. 1977. Isolation of a multienzyme complex of fatty acid
oxidation from Escherichia coli. Proc Natl Acad Sci USA 74: 492-495.
Objective (s) of the Study:
Key Terms
1. -oxidation: a repetitive sequence of four chemical reactions leading to the conversion of long chain fatty acylCoAs to acetyl-CoAs.
2. Disc gel electrophoresis: a technique for separating proteins based on electrophoresis in non-denaturing
acrylamide gels containing a discontinuous buffer system.
3. Phosphocellulose chromatography: a technique for separating proteins based on their affinity for a negativelycharged resin that has been poured into a chromatography column.
4. Sepharose gel filtration chromatography: a technique for separating proteins based on their molecular size
using a resin that has been poured into a chromatography column.
5. Extent of purification: the increase in specific activity that occurs during enzyme purification due to the removal
of proteins lacking the activity of interest.
6. Yield: the amount of enzyme activity that is recovered at the end of a purification procedure relative to the
activity in the crude extract.

Questions
1. What is the function of the -oxidation pathway in biological systems? Write a balanced equation for
the conversion of palmitoyl-CoA to acetyl-coA.
2. What was the evidence that the genes for the enzymes involved in -oxidation in E. coli might be
organized into a functional unit?
3. How was the activity of each of the enzymes in -oxidation sequence measured?
4. What were the steps used to purify the enzymes of -oxidation from E. coli? What was the purpose
of each step?
5. What were the results of subjecting the heat-treated homogenate to phosphocellulose
chromatography? Why did these results support the existence of a multienzyme complex? What might
you have expected to see if the enzymes of -oxidation had not been associated?
6. How did the extents of purification and the yields vary for the thiolase, enoyl-coA hydratase, and hydroxyacyl-coA dehydrogenase activities? How did the authors explain these variations?
7. What was done to show the association of the three enzyme activities was not the result of heat
treatment?
8. What might have happened to the acyl-CoA synthetase and acyl-CoA dehydrogenase activities?
9. What results were obtained when the purified complex was subjected to disc gel electrophoresis and
Sepharose 6B gel filtration chromatography? Why do these results support the existence of a
multienzyme complex?
10. What was the apparent molecular mass of the complex? Why might different techniques give
different values for this molecular mass?
11. How was the subunit structure of the complex determined? What were the results ? How can you
explain the occurrence of three activities and two polypeptides?
12. What was the purpose of the substrate specificity experiment? What were the results?