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Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.

The amino acids are ions of various charge combinations, and one can argue that historically they were the first ions for which the ongoing problem of membrane transport was presented; also that among transported ions these may undergo a highly detailed molecular recognition. Furthermore, the distribution of charge on the amino acid molecule determines by what route or routes it is conducted across the biological membrane, with what directional and structural specificity, and therefore what regulation is imposed, and where. Cases where a presumably charged chemical group behaves as if it were somehow absent from the amino acid have been observed to fall into several categories: Straightforward cases where the pH has been low enough or high enough to remove the charge by protonation or deprotonation, even in free solution. Cases where that protonation or deprotonation is facilitated at the binding site, and perhaps by the total transport process. The cystine molecule can apparently thus be rendered either a tripolar anion or a tripolar cation for transport. Cases where an otherwise co-transported Na+ is omitted to redress charge, or where a Na+ serves as a surrogate for a missing charged group on the amino acid molecule. A case where the protonation occurs reversibly at the receptor site rather than on the amino acid molecule.

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What a strange field is transport! Here the investigator gathers information from kidney tubules, tumor cells, frog skins, gut sacs, and toad bladders. With such as these he observes behavior that violates the intuition of the enzymologist, morphologist, and chemist, and observes the behavior of molecules that may have proved inert in his every other test. Biological transport is by no means a new subject, but it is one that has gained tremendously in interest from the various biological sciences in the last few years. The subject evolved from two rather discrete interests-that of permeability phenomena, extending back more than 60 years, and that of problems of secretion. The difficulties posed by the latter were found in the late 1930s to be associated with the former. With the closer approach to the interpretation of other cellular phenomena, the need to understand how substances and reactions are segregated and brought together in the cell and in the organism has become so pressing that many ingenious, indirect approaches to these questions have been discovered. Meanwhile, the search for the means to identify directly the structures producing transport has continued. This presentation grew from a short series of lectures to an advanced biochemistry class, attended also by graduate students of pharmacology, physiology, microbiology, genetics, and other sciences. It should be interpreted more as a bibliographed syllabus for that instruction than as a review. Accordingly, the author has PREFACE selected the illustrative material and examples that seemed most suitable and familiar to him. The result can hardly seem to have the ideal balance to all the scientific areas now interested in transport. Perhaps physiologists and pharmacologists would like to read more about the intricacies of ion transport, the physical and mathematical biologists more of the theoretical background, the cytolooists more about the morphological substratum, and so on. Although all divergent needs cannot be met, I trust this summary will prove useful to both teaching and learning in various disciplines.