Life in the Open Ocean. Joseph J. Torres

indistinguishable from those of the northern temperate species S. argentea. Error bars represent standard deviations. Each point represents an average of K_m values determined with at least three different purified LDH preparations from different individuals of each species. Solid portions of the lines connecting the K_m values indicate habitat temperature ranges of the species.

      Source: Graves and Somero (1982), figure 3 (p. 103). Reproduced with the permission of John Wiley & Sons, Inc.

Table 2.1 K_m and K_cat values of pyruvate for three congeneric species of barracudas (genus Sphyraena) from different habitat temperatures at 25 °C and the temperature mid‐range (TM) for each species. S. argentea is a cold‐temperate species, S. lucasana is a warm‐temperate species, and S. ensis is a tropical‐subtropical species.

      Source: Graves and Somero (1982), table 5 (p. 104). Reproduced with the permission of John Wiley & Sons, Inc.

	S. argentea	S. lucasana	S. ensis
Km of pyruvate at 25 °C	0.34 ± 0.03 mM	0.26 ± 0.02 mM	0.20 ± 0.02 mM
Kcat at 25 °C	893 ± 54 s^−1	730 ± 37 s^−1	658 ± 19 s^−1
Temp. midrange (TM)	18 °C	23 °C	26 °C
Km of pyruvate at TM	0.24 mM	0.24 mM	0.23 mM
Kcat at TM	667 s^−1	682 s^−1	700 s^−1

      What Properties of Enzymes Can Be Changed?

The three curves in Figure 2.11 show Michaelis–Menten saturation kinetics, exhibiting three very different relationships between reaction velocity and substrate concentration. They illustrate the balance between the need for efficient catalysis and the need for the cell to be able to regulate its metabolism.

      Curve A has a low K_m value; it will always be at or near V_max. Any need for an increase in activity to support increased metabolic demand cannot be met; the enzyme is already at maximum. A low K_m is a fine strategy for an enzyme that does not need to be regulated, such as a digestive enzyme, which is best always functioning at maximum velocity. However, for an enzyme involved in metabolism, which varies from a resting to a highly active state, such a curve would be disastrous. Increases in substrate concentration would not affect its activity, and it would be unable to be regulated. Conversely, a high K_m such as that in curve C will have a considerable amount of “reserve capacity” to allow for regulation but will never achieve high velocity and could become a “choke point” for accumulation of metabolites. An enzyme having an intermediate K_m, curve B, not only has a substantial fraction of its V_max at cellular concentrations of substrate but also has considerable ability to respond to increases in substrate concentration before it reaches V_max. Conclusion: for optimal performance, the enzyme properties and the substrate concentrations available to the enzyme must be complementary.

How variable are physiological substrate concentrations? The answer is, not very: physiological substrate concentrations are highly conserved. Figure 2.12 shows the K_m of pyruvate in LDH from vertebrate species of a wide variety of taxa and habitat temperatures. All K_ms fall between 0.15 and 0.35 mM. Thus, a change in K_m does not appear to be an option in the possibilities available for modification to accommodate temperature change.

Figure 2.11 Michaelis–Menten saturation kinetics: three types of relationships between reaction velocity and substrate concentration. In (A) the K_m value is low and always at or near V_max so that the need for an increase in activity to support increased metabolic demand cannot be met. In (B), the K_m value is intermediate and a substantial fraction of its V_max is at cellular concentrations of substrate giving it considerable ability to respond to increases in substrate concentration before it reaches V_max. In (C), the K_m is high with a considerable amount of “reserve capacity” to allow for regulation but without the ability to ever achieve high velocity and thus the potential to become a “choke point” for accumulation of metabolites.

What other properties of an enzyme can be modified if K_m is not an option? Consider K_cat, the rate at which substrate is converted to product per unit time per active site. If we look at a plot of K_cat vs. body temperature in a large suite of differently thermally adapted vertebrate species (Figure 2.13), it is clear that K_cat declines considerably with increasing species’ habitat temperature. Large differences exist between the K_cat of highly cold‐adapted species and warm‐adapted ectotherms and endotherms. So, unlike K_m, which is highly conserved, we do see large changes with adaptation temperature in K_cat, enzyme efficiency.

Let us now consider how enzymes function. The three‐dimensional conformation of an enzyme and its overall primary structure are somewhat variable between species. However, the structure of the binding sites within the active site, or catalytic vacuole, is highly conserved (Hochachka and Somero 2002). Further, the actual covalent chemistry that takes place during the enzymatic reaction is fast and likely not the limiting step in the turnover of enzyme. The limiting step is more likely related to conformational changes. How do we reconcile the facts that binding sites in the A₄ – LDHs are invariant from species to species, yet we observe a high degree of variability in K_cats? The answer is in changes of structure outside the catalytic vacuole that confer more conformational flexibility at lower temperature. It is believed that enzymes exist in the cell in a hierarchy or ensemble of conformational states, basically varying between binding‐competent and binding‐incompetent (fluctuating) modes. The higher the temperature, the more structurally stable an enzyme has to be to allow the substrate to bind. The tradeoff between efficiency and stability is what results in the trends in K_cat vs. temperature shown in Figure 2.13.

Figure 2.12 Effects of assay temperature on the apparent Michaelis