PART 1 Introduction
4.9. APPENDIX: EXAMPLES OF REGULATION OF COMPLEX PATHWAYS †
In E. colithe conversion of aspartic acid to aspartyl phosphate is mediated by three iso- functional enzymes, of which two (designated as aand cin Fig. 4A.1) also mediate the conversion of aspartic acid semialdehyde to homoserine. Enzyme a, possessing both these functions, is feedback inhibited, and its synthesis is repressed by threonine. Enzyme c, which similarly possesses both functions, is inhibited, and repressed by lysine. The third, aspartokinase (enzyme b), is not subject to end-product inhibition, but its synthesis is re- pressed by methionine (Table 4A.1).
The enzymes of the L-lysine branch (m–q) and the L-methionine branch (r–v) cat- alyze reactions leading in each case to a single end product and are subject to specific re- pression by that end product (L-lysine and L-methionine, respectively).
The third branch of the aspartate pathway is subject to much more complex regula- tion, for two reasons. First, L-threonine, formed through this branch, is both a component of proteins and an intermediate in the synthesis of another amino acid, L-isoleucine. Sec- ond, four of the five enzymes (e–h) that catalyze L-isoleucine synthesis from L-threonine, also catalyze analogous steps in the completely separate biosynthetic pathway by which
L-valine is synthesized from pyruvic acid. The intermediate of this latter pathway, a-ketoisovaleric acid, is also a precursor of the amino acid L-leucine. These interrelation- ships are shown in Fig. 4A.1(b).
L-isoleucine is an end-product inhibitor of the enzyme d, catalyzing the first step in its synthesis from L-threonine; this enzyme has no other biosynthetic role. L-valine is an end- product inhibitor of an enzyme (e) that has a dual metabolic role, since it catalyzes steps in both isoleucine and valine biosynthesis. In certain strains of E. coli, this enzyme is extremely sensitive to valine inhibition, with the result that exogenous valine prevents growth, an effect that can be reversed by the simultaneous provision of exogenous isoleucine. The L-leucine branch of the valine pathway is regulated by L-leucine, which is an end-product inhibitor of the first enzyme, i, specific to this branch. These interrelationships are shown in Fig. 4A.1(b).
As shown in Table 4A.2, many of the enzymes that catalyze steps in the synthesis of
L-isoleucine, L-valine, and L-leucine are subject to repression only by a mixture of the three end products, a phenomenon known as multivalent repression. However, the five en- zymes specific to L-leucine synthesis are specifically repressed by this amino acid alone.
†With permission, from R. Y. Stanier and others, The Microbial World, 5th ed., Pearson Education, Upper Saddle River, NJ, 1986.
TABLE 4A.1 Control of the First Step of the Aspartate Pathway, Mediated by Three Different Aspartokinases, in the Bacterium Escherichia coli
Enzyme Corepressor Allosteric inhibitor
Aspartokinase I Threonine and isoleucine Threonine
Aspartokinase II Methionine No allosteric control
Aspartokinase III Lysine Lysine
Figure 4A.1 A simplified diagram of the aspartate pathway in E. coli. Each solid arrow designates a reaction catalyzed by one enzyme. The biosynthetic products of the pathway (in boldface) are all allosteric inhibitors of one or more reactions. Careful study of this di- agram reveals that with a single exception (the inhibition exerted by valine) the inhibition imposed by one amino acid does not cause starvation for a different amino acid. Part (a) shows the regulatory interrelationships of the L-lysine, L-methionine, and L-isoleucine branches of the pathway. Part (b) shows the regulatory interrelationships of the
L-isoleucine, L-valine, and L-leucine branches
130 How Cells Work Chap. 4
S U G G E S T I O N S F O R F U R T H E R R E A D I N G
ALBERTS, B.,AND OTHERS,Essential Cell Biology,Garland Publishing, Inc., New York, 1998. (This book is an up-to-date, highly readable, relatively short text. The same authors have written a more detailed text, Molecular Biology of the Cell.)
BLACK, J. G.,Microbiology: Principles and Applications3d ed., Prentice Hall, Upper Saddle River, NJ, 1996.
COOK, P. R., The Organization of Replication and Transcription, Science284:1790–1795, 1999.
ELGARD, L., M. MOLINARI,ANDA. HELENIUS, Setting the Standards: Quality Control in the Secre- tory Pathway, Science286:1882–1888, 1999. (Also two related articles on quality control in post- translational processing and translation follow.)
KELLY, M. T., AND T. R. HOOVER, Bacterial Enhancers Function at a Distance, ASM News 65:484–489, 1999.
KOLTER, R., ANDR. LOSICK, One for All and All for One, Science280:226–227, 1998. (This article is an overview related to a more detailed article on quorum sensing in biofilms; see DAVIES, D. G., AND OTHERS,Science280:295–298, 1998.
MADIGAN, M. T., J. M. MARTINKO,ANDJ. PARKER, Brock Biology of Microorganisms,8th ed., Pren- tice Hall, Upper Saddle River, NJ, 1997.
MORAN, L. A., K. G. SCRIMGEOUR, H. R. HORTON, R. S. OCHS,ANDJ. D. ROWN,Biochemistry,2d ed., Prentice Hall, Upper Saddle River, NJ, 1994.
STANIER, R. Y., AND OTHERS,The Microbial World, 5th ed., Prentice Hall, Englewood Cliffs, NJ, 1986.
VONHIPPEL, P. H., An Integrated Model of the Transcription Complex in Elongation, Termination and Editing, Science281:660–665, 1998.
P R O B L E M S
4.1. Consider the aspartic acid pathway shown in Fig. 4A.1. Assume you have been asked to de- velop a high-lysine-producing mutant. What strategy would you pursue? (That is, which steps would you modify by removing feedback inhibition, and what changes in medium composi- tion would you make over a simple mineral salts–glucose base medium?)
4.2. Why is m-RNA so unstable in most bacteria (half-life of about 1 min)? In many higher organ- isms, m-RNA half-lives are much longer (>1 h). Why?
4.3. What would be the consequence of one base deletion at the beginning of the message for a protein?
TABLE 4A.2 Repressive Control of the Enzymes of the Isoleucine—Valine—Leucine Pathway (See Fig. 4A.1)
Enzymes Corepressor
d, e, f, g, h Isoleucine +valine +leucine
i, j, jj, k, l Leucine
4.4. How many ribosomes are actively synthesizing proteins at any instant in an E. colicell grow- ing with a 45-min doubling time? The birth size of E. coliis 1-mm diameter and 2-mm length.
The water content is 75%. About 60%of the dry material is protein, and the rate of amino acid addition per ribosome is 20 amino acids per second. The average molecular weight of free amino acids in E. coliis 126.
4.5. Describe simple experiments to determine if the uptake of a nutrient is by passive diffusion, facilitated diffusion, active transport, or group translocation.
4.6. For the m-RNA nucleotide code below: (a) Deduce the corresponding sequence of amino acids. (b) What is the corresponding nucleotide sequence on the chromosome? This sequence codes for a part of insulin.
CCG UAU CGA CUU GUA ACA ACG CGC
4.7. Consider the pathway in Fig. 4A.1 for production of lysine, methionine, isoleucine, and theronine. You need to produce lysine. Describe a strategy for making large amounts of ly- sine. Your strategy can consist of adding various amino acids to the medium and choosing the mutant cells altered in regulation. Say you can identify up to two points of mutation (e.g., re- moval of feedback inhibition).
4.8. Suggest an experiment to determine if the uptake of a compound is by either facilitated or ac- tive transport.
4.9. What is catabolite repression, and how does it affect the level of protein expression from the lac operon?
4.10. Explain the difference between feedback inhibition and feedback repression.
4.11. You are asked by your boss to produce a human protein in E. coli. Because you have learned some of the differences in the way that procaryotes and eucaryotes make proteins, you worry about at least two factors that could complicate production of an authentic protein for human use.
a) What complication might you worry about if the human DNA encoding the protein were placed directly in E. coli?
b) Assume that the correct primary sequence of amino acids has been produced. What post- translational steps do you worry about and why?
4.12. Consider the process of N-linked glycosylation.
a) What organelles are required?
b) What is the residual sugar on a glycoprotein that has simple glycosylation?
c) If glycosylation is complete, what will be the final sugar on the glycoform?
d) Why may N-linked glycosylation be important?
132 How Cells Work Chap. 4