Over the years, several textbooks on cholinesterases have become widely accepted and used as references in this field. Dr. Ann Silver’s comprehensive book Biology of Cholinesterases, published in 1974, and Drs. Norman Aldridge’s and Elsa Reiner’s elegant text Enzyme Inhibitors as Substrates, with focus on enzyme kinetics, published in 1972, are prime examples. The Moralev’s and Rozengart’s Comparative Enzymology of Cholinesterases should join this list of comprehensive treatises and fill the interdisciplinary gap between the integrated biology of cholinesterase actions and their kinetic mechanisms. Herein, presented for the first time in English language, is more than half century of compiled efforts of scientists from countries of the former USSR. It covers structural and mechanistic data on cholinesterase enzymes from a wide variety of living species and their interactions with a diverse and original collection of substrate and inhibitor compounds. Not only was this wealth of knowledge over many years largely inaccessible for nonslavic language readership through primary scientific publications, but also conference communications on this subject were relatively scarce. Even at the specialized series of International Meetings on Cholinesterases initiated in 1975 in Split, Yugoslavia, and then held over years in Bled, Yugoslavia; Montpellier, France; Eilat, Israel; Madras, India; La Jolla, USA; Pucon, Chile; and Perugia, Italy contained limited data coming from the former Soviet Union. This book serves an important function in making accessible to the Western world valuable data our colleagues in the Soviet Union have generated. We invite you now to peruse this trove of cholinesterase information. We appreciate Professors Moralev and Rozengart’s efforts in assembling the information and developing the English text. We also thank Dr. Igor Tsigelny for recognizing the importance of this topic and editing it for the Biotechnology Series.

At the end of XX century, Brestkin and his co-workers published, in Russian, a book dealing with the comparative enzymology of the cholinesterases of terrestrial animals and hydrobionts (squid species), with special reference to their inhibition by organophosphorus compounds.19 This book summarized more than 40 years of research on catalytic properties and inhibition of cholinesterases, mostly performed by the prestigious Bretskin School of Biochemistry in the Sechenov Institute of Evolutionary Physiology and Biochemistry in Leningrad; the Kabachnik Laboratory in Moscow, which was devoted to the chemistry of organophosphorus compounds; and the group of Jarv and Aaviksaar at the University of Tartu (Estonia). During the Cold-War era, concerned scientists were aware of the efforts made by Soviet chemists and biochemists in the fields of cholinesterase enzymology and organophosphate chemistry since the end of World War II. In particular, an important paper published in 1970 by Kabachnik et al. in Pharmacological Review¹³ revealed the quality of the Soviet basic research on structure-activity relationships of cholinesterases. This landmark paper accurately described the topology of hydrophobic areas of the “active surface” of cholinesterases.¹³ Though the English translation of Biokhimyia was already available in the sixties,^* most of the Soviet works were published in Russian or Ukrainian journals that were difficult to access by non-Slavic scientists. Thus a large part of the Soviet contribution to the biochemistry and enzymology of cholinesterases was underestimated if not ignored.
     During the XIth Symposium on Cholinergic Mechanisms held in Saint Moritz, Switzerland, Serge N. Moralev acknowleged us with the Brestkin’s at al. book19 and the latest results in this area obtained in Russia. The idea of publishing an English book based on the summary of scientific resuts in the area of comparative enzymology of choliesterases, especially describing Soviet and Russian works mostly unknown to the Western reader, came from discussions with P. Taylor and Z. Radič, when it appeared, after reading, that there is a real mine of information, which is still of valuable interest to the scientific community. The present book written by S. N. Moralev and E. V. Rozengart is not an updated translation of the Russian edition of Brestkin’s et al. book. The text was completely recast, and the purpose of the book—mostly focused on the comparative study of the substrate–inhibitor specificity of cholinesterases—presents also new research approaches of the former Bretskin laboratory such as the use of conformational analysis in organophosphate inhibition studies as well as cluster and factor analyses in biochemical taxonomy and phylogeny.

     The first part of the book deals with classical aspects of cholin-esterase specificity in substrate hydrolysis, inhibition by reversible inhibitors, and progressive (“irreversible”) inhibition by organophosphorus compounds and carbamates. The mechanisms of substrate hydro-lysis (esters vs. thioesters), inhibition and/or activation by excess substrate as well as the mechanisms of reversible and irreversible inhibition of cholinesterases by different types of inhibitors are carefully introduced. Sophisticated inhibition studies are described as well, e.g., combined reversible and irreversible inhibition and oxime-mediated reactivation of phosphylated enzymes. The authors systematically explore the effects of length and branching of the alkyl/aryl chains of substrates and inhibitors on enzyme kinetic parameters, using correlation analysis methods. Finally, by combining different approaches, the topography mapping of the different components of the enzyme’s active center can be refined. The numerous tables of catalytic and inhibition parameters compose a veritable treasure chest, a superb and unique database.

     The second part of the book deals with the application of sub-strate–inhibitor specificity for solving some practical problems: (a) calculation of inhibitor selectivity toward different enzymes for identification of reversible and irreversible inhibitors; and (b) the use of specificity constants for molecular phylogenetic analysis of the active center of cholinesterases and esterases. Comparison of computerized dendograms with phylogenetic trees provides evolutionary and taxonomic information that can lead to readjustments in the enzyme nomenclature.

     The authors have to be congratulated for their comprehensive synthesis of a considerable body of scattered works. This book has to be regarded as a reference book and a source of information. We hope that it will be useful to students entering the field of cholinesterase research, and that it will be a valuable guide for enzymologists, molecular pharmacologists, and toxicologists aware of the usefulness of experimental approaches in rational drug research and enzyme engineering.

Year 2004 marked 90 years of research on cholinesterases (ChE). It was in 1914 when Sir Henry Dale while studying the physiological activity of acetylcholine (ACh) and having noted its instability in vivo proposed an enzymatic nature of its hydrolysis.¹ Twelve years afterwards, Loewi and Navratil described for the first time the enzymatic process of ACh hydrolysis in tissues and established simultaneously the fact of inhibition of this process by an alkaloid—eserine.² Thus, although the enzyme was not yet named, its first inhibitor was already discovered. It was only in 1932 that Stedman and co-workers isolated from horse blood serum the enzyme called by them “choline esterase,” which they concluded was as the specific esterase responsible for hydrolysis of ACh and other choline esters.¹ So the enzymologic “christening” of cholinesterase came to pass and four years afterwards the Nobel Prize was awarded to Dale and Loewi for their classical study of ChE as the key enzyme involved in the chemical process of nerve impulse transmission. 
     In the same time, in 1932, Lange and Krueger synthesized the first organophosphorus compounds (OPCs) with a P–F bonds. They experienced the toxic effects of OPCs themselves and even described them.³ Thus, the age of highly toxic OPCs began. In Germany intensive synthesis of organophosphorus poisons was carried out by a team of chemists led by Dr. Gerhard Schrader—Industrial production was organized at that time with an aim for preparation for chemical war.⁴ Similar studies were carried out in England, USA, and in the USSR. In the Soviet Union the chemistry of OPCs was extensively studied in Kazan scientific school directed by A. E. Arbuzov and Moscow scientific school of organoelement compounds led by A. N. Nesmeyanov and M. I. Kabachnik.^4-6 Name of Arbuzov is associated with the basic synthetic methods in OPC chemistry (“Arbuzov’s rearrangement”).
     The association between the toxic OPCs and ChE were discovered only in the beginning of the 1940s. During the war, the majority of academic institutions were evacuated from Moscow and Leningrad to Kazan where A. G. Ginetsinskiy, one of the well-known Russian physiologists, an expert in function of cholinergic systems, focused his research on organophosphorus poisons. Studying a great number of OPCs synthesized for the nonce by M. I. Kabachnik, Ginetsinskiy realized that the complete inhibition of a key enzyme of cholinergic transmission—acetylcholinesterase—is caused by OPC action.⁵ It worth to recall here a tragic episode in this OPCs study. A. G. Ginetsinskiy working in Kazan laboratory in 1942 had spilled an OPC preparation and temporarily lost his vision⁷ (the myotic action is one of typical attributes of heavy OPC poisoning).8 A few decades afterwards, scientific community discovered that several groups of researchers, in different countries, came almost simultaneously to the similar conclusion about OPCs action mechanism, being isolated from each other by national boundaries, lines of fronts of the Second World War, and the dense curtain of secrecy.⁴
     The significance of A. G. Ginetsinskiy’s discovery is difficult to overestimate, since it was broadly applicable, not being only important for military toxicology. First, it made it possible to relate OPC structure to anticholinesterase action and to carry out the directed synthesis of effective inhibitors of ChE (organophosphorus inhibitors—OPIs). Second, testing ChE in various animals, researchers had an opportunity to forecast and obtain effectors with selective toxicity. The new age of organophosphorus insecticides and pharmaceuticals thus began. Third, it was a new stage in research of ChE itself. Using labeled OPIs, amino acid composition of the enzyme active center was revealed. The design of highly specific inhibitors stimulated kinetic studies of the cholinesterase catalysis. New tools became available for comparative research of properties of ChEs in various animals that are in different steps of evolutionary development. The fact that ChE is the target of OPI action has attracted much attention of synthetic chemists, physical chemists, biochemists, pharmacologists, and toxicologists.
     A. G. Ginetsinskiy’s discovery was applied broadly in work by other Russian scientists. For example, V. I. Rozengart was one of the first biochemists in our country actively investigating direct action of OPI on ChE. V. A. Yakovlev made extremely important discovery having revealed that the inhibiting effect of OPIs is caused by phosphorylation of the ChE active center. M.Ya. Mikhel’son was one of the pioneers in the pharmacology of the toxic action of OPIs. A. P. Brestkin developed the kinetic bases of cholinesterase catalysis and interaction with OPIs. All these scientists worked afterwards in the I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry (St. Petersburg), which established a leading role in the development of domestic research of cholinesterases.
     Investigations of the catalytic properties of cholinesterases were carried out in our Institute in several laboratories headed in different periods by Professors V. A. Yakovlev, A. P. Brestkin, V. I. Rozengart, and A. E. Khovanskikh. More than 50 persons participated in different stages of this work.
These researches had two singularities: first, a broad set of topics, and second, extensive cooperation both with physiologists of our Institute and with researchers from other institutes through out the former Soviet Union—biochemists, pharmacologists, toxicologists, entomotoxicologists, and synthetic chemists.

     Three directions of research have been selected:
    1.  Research of the kinetics of ChE interaction with various ligands. These studies were initiated by V. A. Yakovlev and A. P. Brestkin, chemists by higher education and experts in the field of chemical and enzymatic kinetics. Under their supervision, most aspects of cholinesterase catalysis—hydrolysis of substrates, inhibition of ChE activity by reversible inhibitors, and process of interaction of ChE with OPIs—were analyzed. Many of the derived kinetic equations, in our opinion, have not lost their significance to date.
Scientists of the department of general chemistry at the Sanitary and Hygiene Medical Institute led by A. P. Brestkin and of Tartu University (Estonia) directed by A. A. Aaviksaar, a member of National Academy of Sciences, Estonia, also participated in this work.
   2.  Research in the catalytic properties of cholinesterases in animals at different levels of evolutionary development. This direction was inured, on the one hand, by the evolutionary and comparative biological profile of our Institute, and, on the other hand, focused by applied research—searching for the bases of directed synthesis of highly effective and selective agents for plant protection. Cholinesterases from more than 60 animal species—vertebrates, arthropods, and mollusks—were studied.
     In our Institute, the comparative research of cholinergic systems in various animals was carried out also at the laboratory of pharmacology (headed first by Professor M. Ya. Mikhel’son, then by Professor A. F. Danilov, and now by Professor N. Ya. Lukomskaya) and at the laboratory of neurophysiology (headed previously by Professor A. K. Voskresenskaya and now by V. L. Sviderskiy, a member of Russian Academy of Sciences). Scientists of the Institute of Plant Protection in St. Petersburg directed by K. V. Novozhilov, a member of Russian Academy of Agricultural Sciences, department of biochemistry of the Turkmenia Medical Institute in Ashkhabad headed by Professor A. M. Kulieva, laboratory of technical biochemistry of the Pacific Institute of Fish Facility and Oceanography in Vladivostok headed by Professor L. M. Epshteyn, and, to a lesser degree, of other institutions participated in comparative studies of ChE in various animals as well.
  3. Research of relationships between structure and activity of various effectors of cholinesterases. Structure-activity relations of a large number of substrates and reversible and irreversible (organophosphorus and carbamate) inhibitors were investigated. The most modest estimates of their total number give more than 1.5 thousands compounds.
     Teams of synthetic chemists from various regions of former Soviet Union perennially collaborated in this work. The laboratories led by a member of Russian Academy of Sciences M. I. Kabachnik, corresponding member of the Russian Academy of Sciences T. A. Mastryukova, and Professor N. N. Godovikov (A. N. Nesmeyanov Institute of Organoelement Compounds, Moscow), a member of Russian Academy of Medical Sciences N. V. Khromov-Borisov (Institute of Experimental Medicine, St. Petersburg), Professors S. G. Kuznetsov and S. M. Ramsh (Institute of Toxicology, St. Peters-burg), Professor N. K. Skvortsov (Technological Institute, St. Petersburg), a member of Russian Academy of Sciences M. G. Voronkov (A. E. Favorskij Institute of Organic Chemistry, Irkutsk, Russia), a member of National Academy of Sciences A. A. Abduvakhabov (A. S. Sadykov Institute of Organic Chemistry, Tashkent, Uzbekistan), Professor L. M. Yagupol’skiy (Institute of Organic Chemistry, Kiev, Ukraine), and a member of National Academy of Sciences A. A. Aaviksaar (Tartu University, Estonia) participated in this integration of chemistry and enzymology. The method of theoretical conformational analysis to study interaction of the ligands with the ChE active center was developed for the first time in our country in our institute in collaboration with colleagues from laboratory of molecular physiology (led by a member of Russian Academy of Sciences V. A. Govyrin, and now by Professor A. I. Krivchenko).
     This direction of research was bound tightly with a comparative enzymology. Many compounds were investigated in reaction of interaction not only with “typical” mammalian ChEs—human erythrocyte AChE and horse blood serum BChE (in collaboration with the department of biochemistry of First I. P. Pavlov Medical Institute at St. Petersburg headed previously by Professor V. I. Rozengart and now by Professor I. G. Shcherbak)—but also with ChEs of other vertebrates as well as with ChEs from various insects (in collaboration with Institute of Plant Protection) and mollusks (in collaboration with Pacific Institute of Fish Facility and Oceanography).

     This large concerted group of like-minded collaborators that was gradually evolved from the end of 1940s worked successfully and fruitfully during more than 40 years. Results of their work were published in many monographs^8-20 and large reviews. Despite our desire, we are not able to cite all of the obtained data within the framework of this book. Therefore we shall condense data in tables, with accompaning brief analysis and discussion from the point of view of the modern data on structure of ChE active center and the mechanism of cholinesterase catalysis.

   The catalytic properties of the mammalian AChE and BChE in reactions with substrates and reversible and irreversible inhibitors of various structure are considered in Chapters 1–4. We also summarized the data on the substrate and inhibitor specificity of ChEs in different animals.
We did our best in such an attempt, but we have to note that, unfortunately, not all appropriate papers in Western journals were available for us).
    In Chapter 5 the theoretical basis and practical examples for identification of irreversible and reversible inhibitors using cholinesterase specificity are presented.
    The Chapter 6 is devoted to discussion about the cholinesterases substrate–inhibitor specificity in a evolutionary aspect.
     We used these numerous data as a “raw material” for statistical processing. Multidimensional methods dominate among current methods of statistical data analysis and allow to cover simultaneously a broad spectrum of parameters and their relations elucidating their basic principal associations. Such a reduced number of parameters is easier for the constructive perception and further analysis than the multidimensional space of initial parameters. This is fully related to many biological characteristics including general “reactivity” of enzymes, which is by its virtue a multidimensional score function and cannot be measured directly.
     We hope that the data summarized in this book and the results obtained by their statistical analysis would be useful for understanding mechanisms of cholinesterase catalysis, for studing comparative enzymology of ChE, and for searching new highly effective and highly selective insecticides and drugs.