A portion of the research was carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission. This work was also supported by the National Science Foundation.

The transient and steady-state behavior of a reversible covalent modification system is examined. When the modifying enzymes operate outside the region of first-order kinetics, small percentage changes in the concentration ofthe effector controlling either ofthe modifying enzymes can give much larger percentage changes in the amount of modified protein. This amplification of the response to a stimulus can provide additional sensitivity in biological control, equivalent to that of allosteric proteins with high Hill coefficients.Biological systems must respond to internal and external variations such as the depletion of nutrients, the variations in hormone levels, and the reception of sensory signals. The stimuli are processed to change the activities of enzymes controlling pathways in the biological system. Two basic phenomena play a large role in this processing: allosteric changes in protein conformation and covalent modification of proteins.Since the findings of Cori and Green (1) and Krebs and Fischer (2) that glycogen phosphorylase exists in two forms, phosphorylated and dephosphorylated, the number ofproteins that have been found to be controlled by covalent modification has increased steadily. Covalent modification has been identified with control in carbohydrate metabolism, fat metabolism, sensory systems, muscular contraction, protein synthesis, nitrogen metabolism, and malignant transformation (3-10).In phenomena such as sensing, and in the regulation of metabolism, it is important that the "turning on" of one pathway and the "turning off" of another be sensitive to relatively small changes in effector concentration. One known mechanism for increasing the sensitivity of a system is through cooperative interactions. Another is the effect of a ligand that enters at more than one step in a pathway-e.g., to activate one enzyme and inhibit another, as happens in the glycogen cascade (4

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A "temporal gradient apparatus" has been developed that allows the motility of bacteria to be studied after they have been subjected to a sudden change from one uniform concentration of attractant to another. A sudden decrease elicits the tumbling response observed with spatial gradients; it was found, however, that a sudden increase also elicits a response, namely supercoordinated swimming. This demonstrates that chemotaxis is achieved by modulation of the incidence of tumbling both above and below its steady-state value. The initial responses gradually revert to the steady-state motility pattern characteristic of a uniform distribution of attractant. The apparent detection of a spatial gradient by the bacteria therefore involves an actual detection of a temporal gradient experienced as a result of movement through space. Potential models for the chemotactic response based on some "memory" mechanism are discussed.The phenomenon of chemotaxis occurs widely in biological systems. Its presence in bacteria was detected by Pfeffer (1) in 1881, and has been clarified further in recent years, in particular by the recent studies of Adler and his coworkers (2, 3). In many ways bacterial chemotaxis appears analogous to sensory reception in higher species as in (a) the specificity of the response to attractants (2, 4), (b) the indication that the receptor molecules are located in the outer membrane (3, 5), and (c) the sensitivity of the response to ratios of concentrations rather than to differences (1, 6).4 However, bacterial chemotaxis poses a special problem: how can such a small organism detect the concentration differences necessary to sense a gradient in space?The "size problem" in relation to gradient sensing can be readily calculated. In an exponential gradient with a decay distance of 20 mm, the difference in concentration of attractant at the two extremes of a 2-utm long bacterium is only 1 part in 104. Since bacteria respond strongly in such a gradient, an analytical device capable of discerning 1 part in 104 is required if the sensing system simply utilizes spatial separation. A further problem arises in relation to the statistical fluctuations of attractant in the vicinity of the receptors.Assuming hypothetical sampling volumes of 1 am X 1 gm X 0.1 um, near the "head" and "tail" of a bacterium, only 60 molecules of attractant would be present at 1 MM, yet chemotaxis is known to occur at such concentrations. The standard deviation of 60 molecules is 1 /60, showing that statistical fluctuations can be much greater than the needed accuracy.

The three-dimensional structure of an ictive, disulfide cross-linked dimer of the ligand-binding domain of the Salmonella typhimurium aspartate receptor and that of an aspartate complex have been determined by x-ray crystallographic methods at 2.4 and 2.0 angstrom (A) resolution, respectively.A single subunit is a four-cy-helix bundle with two long amino-terminal and carboxyl-terminal helices and two shorter helices that form a cylinder 20 A in diameter and more than 70 A long. The two subunits in the disade-bonded dimer are related by a crystallographic twofold axis in the apo structure, but by a noncrystallographic twofold axis in the aspartate complex structure.

A startlingly simple molecule unites neuroscience, physiology, and immunology, and revises scientists' understanding of how cells communicate and defend themselves.

It is a great pleasure for me to contribute to this symposium honoring the great scientist Emil Fischer. My graduate thesis required me to synthesize [I -'4C]glucose, which introduced me to the famous Fischer-Kiliani synthesis of glucose and mannose from arabinose and HCN. ['] I was also particularly intrigued with his classic key-lock (or template) theory of enzyme specificity,12. 31 which like all great theories seemed so obvious once one understood it.This symposium in his honor allows me to pay tribute to Fischer's great contributions to biochemistry varying from natural products chemistry to the key-lock theory, to review some of the history and significance of our induced fit theory, to illustrate the ramifications of those theories in our present era of protein -1igand interactions, and to discuss recent work in our laboratory which is helping to clarify conformational changes and their function. These theories have assumed again a central role in modern health research where the need for drug design requires taking into account the complementarity of fit of Fischer's principle and the flexibility and regulatory implications of the induced fit theory.The induced fit theory is no more a refutation of Fischer's key-lock principle than the Heisenberg atom was of the Bohr atom or the modern D N A sequences are of the one gene-one enzyme hypothesis. A new theory must explain all the existing facts that pertain to it at the time of its enunciation. Gradually the new theory becomes accepted and then acquires anomalies due to the new facts uncovered after its enunciation. That in turn generates a newer theory which elicits new techniques to test it and its predictions. These new techniques then uncover facts which eventually require further new theories and so on. The new theories are built on components of the old principles. It is said that each scientist stands on the shoulders of the giants who have gone before him. There can be no more honored place than to stand on the shoulders of Emil Fischer.water in biological reactions. I was preparing a lecture for a scientific meeting and decided to consider why some proteins were kinases and others ATPases. The more I thought about the protein, the more astonishing it seemed that water could be prevented from reacting at the active site of a kinase.In hexokinase, which I took as a typical kinase, the O H group of water was known to be as good a nucleophile as the OH group of a sugar. If glucose is bound very tightly then it could exclude water, and a basic group on the protein would generate a glucosyl oxyanion nucleophile which could attack the ATP. But glucose would not normally saturate the site and could in many physiological circumstances fall to very low levels. Water, at 55 M, would fill up an empty site, and therefore water would be constantly competing with glucose in the nucleophilic attack on ATP. The existence of kinases in the absence of substrate or with only partially filled template type active sites would result in great ATPase activity and an enorm...

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