PART 1 Introduction
4.7. HOW THE CELL SENSES ITS EXTRACELLULAR ENVIRONMENT 1. Mechanisms to Transport Small Molecules
isozymes (E2), while the other enzyme (E¢2) is fully active. Sufficient activity remains through isozyme E¢2to ensure adequate synthesis of P2.
An alternative approach is concerted feedback inhibition. Here a single enzyme with two allosteric binding sites (for P1and P2) controls entry into the pathway. A high level of either P1or P2is not sufficient by itself to inhibit enzyme E2, while a high level of both P1and P2will result in full inhibition.
A third possibility is sequential feedback inhibition,by which an intermediate at the branch point can accumulate and act as the inhibitor of metabolic flux into the pathway.
High levels of P1 and P2inhibit enzymes E4 and E5, respectively. If either E4 or E5 is blocked, M3will accumulate, but not as rapidly as when both E4or E5are blocked. Thus, intermediate flux levels are allowed if either P1or P2is high, but the pathway is inacti- vated if both P1and P2are high.
Other effects are possible in more complex pathways. A single allosteric enzyme may have effector sites for several end products of a pathway; each effector causes only partial inhibition. Full inhibition is a cumulative effect, and such control is called cumula- tive feedback inhibitionor cooperative feedback inhibition. In other cases, effectors from related pathways may also act as activators. Typically, this situation occurs when the prod- uct of one pathway was the substrate for another pathway. An example of control of a complex pathway (for aspartate) is described in Section 4.9.
The reader should pause to consider the differences between feedback inhibition and repression. Inhibition occurs at the enzyme level and is rapid; repression occurs at the genetic level and is slower and more difficult to reverse. In bacteria where growth rates are high, unwanted enzymes are diluted out by growth. Would such a strategy work for higher cells in differentiated structures? Clearly not, since growth rates would be nearly zero. In higher cells (animals and plants) the control of enzyme levels is done primarily through the control of protein degradation, rather than at the level of synthesis. Most of our discussion has centered on procaryotes; the extension of these concepts to higher or- ganisms must be done carefully.
Another caution is that the control strategy that one organism adopts for a particular pathway may differ greatly from that adopted by even a closely related organism with an identical pathway. Even if an industrial organism is closely related to a well-studied or- ganism, it is prudent to check whether the same regulatory strategy has been adopted by both organisms. Knowing the cellular regulatory strategy facilitates choosing optimal fer- menter operating strategy, as well as guiding strain improvement programs.
We have touched on some aspects of cellular metabolic regulation. A related form of regulation that we are just now beginning to appreciate has to do with the cell surface.
4.7. HOW THE CELL SENSES ITS EXTRACELLULAR ENVIRONMENT
Molecules enter the cell through either energy-independent or energy-dependent mechanisms. The two primary examples of energy-independent uptake are passive diffu- sionand facilitated diffusion. Energy-dependent uptake mechanisms include active trans- portand group translocation.
In passive diffusion, molecules move down a concentration gradient (from high to low concentration) that is thermodynamically favorable. Consequently,
(4.1) where JAis the flux of species Aacross the membrane (mol/cm2- s), Kpis the permeabil- ity (cm/s), CAEis the extracellular concentration of species A(mol/cm3), and CAIis the in- tracellular concentration. The cytoplasmic membrane consists of a lipid core with perhaps very small pores. For charged or large molecules, the value Kpis very low and the flow of material across the membrane is negligible. The cellular uptake of water and oxygen ap- pears to be due to passive diffusion. Furthermore, lipids or other highly hydrophobic com- pounds have relatively high diffusivities (10-8cm2/s) in cellular membranes, and passive diffusion can be a mechanism of quantitative importance in their transport.
With facilitated transport, a carrier molecule (protein) can combine specifically and reversibly with the molecule of interest. The carrier protein is considered embedded in the membrane. By mechanisms that are not yet understood, the carrier protein, after binding the target molecule, undergoes conformational changes, which result in release of the molecule on the intracellular side of the membrane. The carrier can bind to the target mol- ecule on the intracellular side of the membrane, resulting in the efflux or exit of the mole- cule from the cell. Thus, the net flux of a molecule depends on its concentration gradient.
The carrier protein effectively increases the solubility of the target molecule in the membrane. Because the binding of the molecule to the carrier protein is saturable (just as the active sites in the enzyme solution can be saturated), the flux rate of the target mole- cule into the cell depends on concentration differently than indicated in eq. 4.1. A simple equation to represent uptake by facilitated transport is
(4.2) where KMTis related to the binding affinity of the substrate (mol/cm3) and JA MAX is the maximum flux rate of A(mol/cm2-s). When CAE>CAI, the net flux will be into the cell. If CAI>CAE, there will be a net efflux of Afrom the cell. The transport is down a concentra- tion gradient and is thermodynamically favorable. Facilitated transport of sugars and other low-molecular-weight organic compounds is common in eucaryotic cells, but infrequent in procaryotes. However, the uptake of glycerol in enteric bacteria (such as E. coli) is a good example of facilitated transport.
Active transport is similar to facilitated transport in that proteins embedded in the cellular membrane are necessary components. The primary difference is that active trans- port occurs against a concentration gradient. The intracellular concentration of a mole- cule may be a hundredfold or more greater than the extracellular concentration. The movement of a molecule up a concentration gradient is thermodynamically unfavorable and will not occur spontaneously; energy must be supplied. In active transport, several en- ergy sources are possible: (1) the electrostatic or pH gradients of the proton-motive force,
J J C
K C
C
K C
A A AE
MT AE
AI
MT AI
= + -
+ È
ÎÍ ˘
˚˙
MAX
JA=K Cp( AE-CAI)
and (2) secondary gradients (for example, of Na+or other ions) derived from the proton- motive force by other active transport systems and by the hydrolysis of ATP.
The proton-motive forceresults from the extrusion of hydrogen as protons. The res- piratory system of cells (see Section 5.4) is configured to ensure the formation of such gradients. Hydrogen atoms, removed from hydrogen carriers (most commonly NADH) on the inside of the membrane, are carried to the outside of the membrane, while the elec- trons removed from these hydrogen atoms return to the cytoplasmic side of the mem- brane. These electrons are passed to a final electron acceptor, such as O2. When O2 is reduced, it combines with H+from the cytoplasm, causing the net formation of OH-on the inside. Because the flow of H+and OH-across the cellular membrane by passive dif- fusion is negligible, the concentration of chemical species cannot equilibrate. This process generates a pH gradient and an electrical potential across the cell. The inside of the cell is alkaline compared to the extracellular compartment. The cytoplasmic side of the mem- brane is electrically negative, and the outside is electrically positive. The proton-motive force is essential to the transport of many species across the membrane, and any defect in the cellular membrane that allows free movement of H+and OH-across the cell boundary can collapse the proton-motive force and lead to cell death.
Some molecules are actively transported into the cell without coupling to the ion gradients generated by the proton-motive force. By a mechanism that is not fully under- stood, the hydrolysis of ATP to release phosphate bond energy is utilized directly in trans- port (e.g., the transport of maltose in E. coli).
For these mechanisms of active transport, irrespective of energy source, we can write an equation analogous to Michaelis–Menten kinetics to describe uptake:
(4.3) The use of eq. 4.3 is meaningful only when the cell is in an energy-sufficient state.
Another energy-dependent approach to the uptake of nutrients is group transloca- tion. The key factor here is the chemical modification of the substrate during the process of transport. The best-studied system of this type is the phosphotransferase system. This system is important in the uptake of many sugars in bacteria. The biological system itself is complex, consisting of four separate phosphate-carrying proteins. The source of energy is phosphoenolpyruvate (PEP).
Effectively, the process can be represented by:
(4.4) By converting the sugar to the phosphorylated form, the sugar is trapped inside the cell. The asymmetric nature of the cellular membrane and this process make the process essentially irreversible. Because the phosphorylation of sugars is a key step in their metabolism, nutri- ent uptake of these compounds by group translocation is energetically preferable to active transport. In active transport, energy would be expended to move the unmodified substrate into the cell, and then further energy would be expended to phosphorylate it.
Certainly, the control of nutrient uptake is a critical cellular interface with its extra- cellular environment. In some cases, however, cells can sense their external environment without the direct uptake of nutrients.
sugar(extracellular) + PEP(intracellular) Æ sugar - P(intracellular) + pyruvate(intracellular)
J J C
K C
A A AE
MT AE
= MAX +
126 How Cells Work Chap. 4
4.7.2. Role of Cell Receptors in Metabolism and Cellular Differentiation
Almost all cells have receptors on their surfaces. These receptors can bind a chemical in the extracellular space. Such receptors are important in providing a cell with information about its environment. Receptors are particularly important in animals in facilitating cell- to-cell communication. Animal cell surface receptors are important in transducing signals for growth or cellular differentiation. These receptors are also prime targets for the devel- opment of therapeutic drugs. Many viruses mimic certain chemicals (e.g., a growth factor) and use cell surface receptors as a means to entering a cell.
Simpler examples exist with bacteria. Some motile bacteria have been observed to move up concentration gradients for nutrients or down gradients of toxic compounds. This response is called chemotaxis. Some microbes also respond to gradients in oxygen (aero- taxis) or light (phototaxis). Such tactic phenomena are only partially understood. How- ever, the mechanism involves receptors binding to specific compounds, and this binding reaction results in changes in the direction of movement of the flagella. Motile cells move in a random-walk fashion; the binding of an attractant extends the length of time the cell moves on a “run” toward the attractant. Similarly, repellents decrease the length of runs up the concentration gradient. Chemotaxis is described in Fig. 4.13.
Figure 4.13. Diagrammatic representation of Escherichia colimovement, as analyzed with the tracking microscope. These draw- ings are two-dimensional projections of the three-dimensional movement. (a) Random movement of a cell in a uniform chemical field. Each run is followed by a twiddle, and the twiddles occur fairly frequently. (b) Di- rected movement toward a chemical attrac- tant. The runs still go off in random directions, but when the run is up the chemi- cal gradient, the twiddles occur less fre- quently. The net result is movement toward the chemical. (c) Directed movement away from a chemical repellent. (With permission, adapted from T. D. Brock, D. W. Smith, and M. T. Madigan, Biology of Microorganisms, 4th ed., Pearson Education, Upper Saddle River, NJ, 1984, p. 43.)
Microbial communities can be highly structured (e.g., biofilms), and cell-to-cell communication is important in the physical structure of the biofilm. Cell-to-cell commu- nication is also important in microbial phenomena such as bioluminescence, exoenzyme synthesis, and virulence factor production. Basically, these phenomena depend on local cell concentration. How do bacteria count? They produce a chemical known as quorum sensing molecule, whose accumulation is related to cell concentration. When the quorum sensing molecule reaches a critical concentration, it activates a response in all of the cells present. A typical quorum sensing molecule is an acylated homoserine lactone. The mech- anism of quorum sensing depends on an intracellular receptor protein, while chemotaxis depends on surface receptor proteins.
With higher cells, the timing of events in cellular differentiation and development is associated with surface receptors. With higher organisms, these receptors are highly evolved. Some receptors respond to steroids (steroid hormone receptors). Steroids do not act by themselves in cells, but rather the hormone–receptor complex interacts with spe- cific gene loci to activate the transcription of a target gene.
A host of other animal receptors respond to a variety of small proteins that act as hormonesor growth factors. These growth factors are normally required for the cell to ini- tiate DNA synthesis and replication. Such factors are a critical component in the large- scale use of animal tissue cultures. Other cell surface receptors are important in the attachment of cells to surfaces. Cell adhesion can lead to changes in cell morphology, which are often critical to animal cell growth and normal physiological function. The exact mechanism by which receptors work is only now starting to emerge. One possibility for growth factors that stimulate cell division is that binding of the growth factor to the re- ceptor causes an alteration in the structure of the receptor. This altered structure possesses catalytic activity (e.g., tyrosine kinase activity), which begins a cascade of reactions lead- ing to cellular division. Surface receptors are continuously internalized, complexes de- graded, and receptors recycled to supplement newly formed receptors. Thus, the ability of cells to respond to changes in environmental signals is continuously renewed. Such recep- tors will be important in our later discussions on animal cell culture.