Harvey Alan Berman

Structural bases for the specificity of cholinesterase catalysis and inhibition.

The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.

Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates.

A series of eight double and triple mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7), with substitutions corresponding to residues found largely within the butyrylcholinesterase (BChE; EC 3.1.1.8) active-centre gorge, was analysed to compare steady-state kinetic constants for substrate turnover and inhibition parameters for enantiomeric methylphosphonate esters. The mutations combined substitutions in the acyl pocket (Phe(295)-->Leu and Phe(297)-->Ile) with the choline-binding site (Tyr(337)-->Ala and Phe(338)-->Ala) and with a side chain (Glu(202)--> Gln) N-terminal to the active-site serine, Ser(203). The mutations affected catalysis by increasing K (m) and decreasing k (cat), but these constants were typically affected by an order of magnitude or less, a relatively small change compared with the catalytic potential of AChE. To analyse the constraints on stereoselective phosphonylation, the mutant enzymes were reacted with a congeneric series of S (P)- and R (P)-methylphosphonates of known absolute stereochemistry. Where possible, the overall reaction rates were deconstructed into the primary constants for formation of the reversible complex and intrinsic phosphonylation. The multiple mutations greatly reduced the reaction rates of the more reactive S (P)-methylphosphonates, whereas the rates of reaction with the R (P)-methylphosphonates were markedly enhanced. With the phosphonates of larger steric bulk, the enhancement of rates for the R (P) enantiomers, coupled with the reduction of the S (P) enantiomers, was sufficient to invert markedly the enantiomeric preference. The sequence of mutations to enlarge the size of the AChE active-centre gorge, resembling in part the more spacious gorge of BChE, did not show an ordered conversion into BChE reactivity as anticipated for a rigid template. Rather, the individual aromatic residues may mutually interact to confer a distinctive stereospecificity pattern towards organophosphates.

Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity.

We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.

Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle

Developmental regulation, from the fetal period to 11 months of age, and the influence of denervation on the appearance and disappearance of the molecular forms of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) in rat skeletal muscle were examined. The enzyme forms were extracted from anterior tibialis in 0.01 M sodium phosphate buffer, pH 7.0, containing 1 N NaCl, 0.01 M EGTA, 1% Triton X-100, and a cocktail of antiproteases, and analyzed by velocity sedimentation on 5-20% linear sucrose gradients. Three principal forms, denoted by sedimentation coefficients of 4, 10.8, and 16 S, were observed in muscle from all age groups. The amounts of each of the molecular forms of AchE and BuchE in skeletal muscle exhibited distinct and reciprocal patterns of appearance and disappearance during pre- and postnatal development. In tissue derived from animals less than 2 weeks of age, BuchE represented the predominant component of activity in the 4 S form, was present equally with AchE in the 10.8 S form, and was subordinate to AchE in the 16 S form. Between 1 and 2 weeks of age a progressive increase in AchE activities coincident with a reduction in BuchE activities resulted in inversion in the amounts of the two enzymes present in adult muscle. Denervation of muscle caused a dramatic reduction in the presence of AchE molecular forms with no discernable influence on the presence of BuchE molecular forms. These results indicate that biosynthesis of BuchE is strictly regulated in a reciprocal manner with that of AchE, and that BuchE metabolism is independent of the state of muscle innervation. Increased synthesis of AchE and either reduced synthesis or increased degradation of BuchE can account for the reciprocal regulation of these enzymes. These characteristics of mammalian muscle contrast sharply with characteristics deduced for avian tissue (Silman et al. (1979) Nature (London) 280, 160-162). The innervation-independent metabolism of BuchE and the diverse modes of its regulation in different tissue from different species signify that BuchE function may be unrelated to cholinergic neurotransmission.

Kinetic, equilibrium and spectroscopic studies on cation association at the active center of acetylcholinesterase: topographic distinction between trimethyl and trimethylammonium sites

This study examines the importance of electrostatic interactions on ligand association at the active center of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). The active-center serine was covalently modified with the dimensionally equivalent isosteric beta-(trimethylammonium)ethyl and 3,3-dimethylbutyl methylphosphonofluoridates. Reactivation of the 3,3-dimethylbutyl methylphosphono-conjugate by the bisquaternary mono-oxime HI-6, after accounting for the capacity for spontaneous reactivation, proceeded at a rate that was 20-fold greater than that for the cationic conjugate. Decidium, a fluorescent bisquaternary ligand that binds with its trimethylammonium moiety within the active center, exhibited affinity for the 3,3-dimethylbutyl conjugate that was within 2-fold that for the native enzyme, but 100-fold greater than for the cationic conjugate. Whereas association of n-alkyl mono- and bisquaternary ligands with the uncharged conjugate was virtually unaltered with respect to the native enzyme, the affinities of edrophonium, phenyltrimethylammonium and N-methylacridinium were reduced 100-fold for the uncharged conjugate relative to native enzyme. These results indicate that the orientations of the 3,3-dimethylbutyl and beta-(trimethylammonium)ethyl moieties with respect to the surface of the enzyme are not equivalent, that modification of the active center does not preclude cation association of active-center-selective ligands, and that aromatic cations associate at an anionic locus which is unique from that at which decidium and the n-alkyl mono- and bisquaternary cations associate. As such, the results point to the presence of a heterogeneity of cation binding sites within a circumscribed distance from the modified serine, and do not sustain the view proposed by Hasan et al. (J. Biol. Chem. 255 (1980) 3898-3904; 256, (1981) 7781-7785) that electrostatic interactions at the active center are subordinate to steric constraints imposed by a dimensionally restricted trimethyl site.

The Tufts partnership for managed care education.

The authors describe the formation and the academic activities of the Tufts Managed Care Institute, a collaborative venture of Tufts University School of Medicine and Tufts Health Plan, an independent-practice-association (IPA)-model health maintenance organization (HMO). In 1994, the dean of the medical school and the CEO of the HMO recognized the need for collaboration to prepare students and practitioners for high-quality, cost-effective practice in a managed care environment. They established an advisory committee to oversee a six-month feasibility study to interview experts and opinion leaders and identify critical characteristics of the ideally prepared managed care practitioner. In 1995, with start-up funding from the HMO, the institute began its operations as a freestanding enterprise with board representation from the two sponsoring institutions. While many of the institutes programs have been developed for practicing physicians and other health care professionals, this article focuses on the academic activities. For medical students, the approach has been to blend managed care principles and practices into existing courses, problem-based learning cases, and clerkships, rather than creating separate managed care courses. For primary care residents, the institute has a grant to develop managed care curricula in conjunction with Tufts-affiliated residency training programs. Faculty development is accomplished through dedicated workshops and seminars, and through increased dialogue between traditional faculty and managed care professionals. In all of its programs and activities the institute has fostered greater contact and collaboration between colleagues from both sides of the health plan-academia “divide.” Operationally, the institute structure, with dedicated full-time administrative staff, provides the singular focus necessary to establish managed care education as a top priority for the partnership. At the same time, sustaining this freestanding organization and infrastructure requires increased resources. Initial responses to the institutes programs and activities have been positive, both from the local Tufts community and from external parties. Yet the partnership must establish methods to evaluate the institutes long-term impact in its efforts to help practitioners succeed in a transforming landscape.

Denervation-induced alterations of acetylcholinesterase in denervated and nondenervated muscle

The influence of denervation on acetylcholinesterase (AchE) molecular forms in rat skeletal muscle for durations up to 30 days is examined in denervated anterior tibialis, the innervated contralateral muscle, and diaphragm. Denervated rats at a common age of 8.5 weeks are compared with age-matched, nondenervated animals. The results indicate that time-dependent losses of AchE in denervated muscle occur more rapidly than loss of muscle mass and are not uniform among the different molecular forms. Loss of the 4 S and 16 S forms is rapid and essentially complete within 3.5 days of denervation, while during this same period the 10.5 S form undergoes a transient twofold increase and its presence in denervated muscle is never abolished. Within 30 days of denervation, all forms of AchE including the 16 S species reappear. A salient finding of these studies is that the effects of denervation are evident also in anatomically remote, innervated muscle such as anterior tibialis of the contralateral limb and in diaphragm. These alterations appear as pronounced reductions in 4 S AchE and increases in 10.5 S AchE; the asymmetric collagen-tailed 16 S form is unaltered. Treatment of primary cultures of embryonic chick pectoral muscle with sera from denervated but not nondenervated rat causes reductions in AchE. These results indicate that the appearance and retention of AchE, in particular the 16 S form, occur in the absence of functional innervation. The effects of denervation on AchE metabolism in remote, innervated tissue are consistent with the action of a diffusible factor released from severed nerve or muscle, or both.

Regulation of acetylcholinesterase in avian heart. Studies on ontogeny and the influence of vagotomy.

This article examines the role of innervation in regulating expression of acetylcholinesterase (AchE), butyrylcholinesterase (BuchE), and the muscarinic acetylcholine receptor (mAchR) in avian heart. Two distinct approaches are taken. The first approach examines the relation between the onsets of parasympathetic and sympathetic innervation and the appearance of AchE and BuchE. All molecular forms of AchE and BuchE are present in early embryonic chick heart well before the onset of parasympathetic and sympathetic innervation. These molecular forms are characterized by sedimentation coefficients of 4.5S, 11S, 15S, and 19S. With further development, the amounts of AchE fall; the reductions in AchE parallel the onset of functional parasympathetic innervation. The amounts of BuchE increase progressively throughout embryonic development, independent of autonomic innervation, and in mature chick heart predominate over the much less abundant amounts of AchE. The 15S and 19S forms of AchE in heart are lost during early embryogenesis but reappear in skeletal muscle during later embryogenesis. The second approach examines the influence of vagotomy and sympathetic denervation of 8-day-old chick myocardium on expression of the molecular forms of AchE, BuchE, mAchR, and beta-adrenergic receptors. The amounts of AchE and BuchE molecular forms in avian heart are not measurably influenced by bilateral vagotomy for a duration of 4 days, unilateral vagotomy for a duration of 25 days, or sympathetic denervation. A measurable upregulation is observed in muscarinic receptors (35-46%) after vagotomy but not sympathectomy and in beta-adrenergic receptors (29%) after sympathectomy but not vagotomy. In all cases, results in atria and ventricles are nearly identical. The present results indicate that expression of AchE in the myocardium is unique and different from that in skeletal muscle and not directly linked with autonomic innervation.

Influence of Ionic Composition of the Medium on Acetylcholinesterase Conformation

The recent crystallographic analysis of a dimeric glycophospholipid form of AchE from Torpedo californica (Sussman et al., 1991) provides an ideal opportunity for comparing the catalytic behaviour and recognition properties of the enzyme with its atomic structure. The globular subunit is determined to be an α/s protein containing numerous crossover motifs of the type s-α-s or s-loop-s. The catalytic residue, Ser 200, resides 4 A above the base of a 20 A deep cavity - or gorge - lined with numerous aromatic amino acid residues accounting for approximately 40 percents of the active center surface. A catalytic charge-relay triad comprising Ser 200-His 440-Glu327 is identified. Quite surprisingly, only few net negative amino acid residues are found within the gorge: Asp 285 and Glu 273 at the top, Asp 72 about half-way into the gorge, and G1u 199, proximal to Ser 200, near the base. Overall, the atomic coordinates afford a picture of the active center as that of a deep, highly aromatic cavity, rich in pi-electron density. No evidence exists for an anionic choline-binding locus situated approximately 5 A from Ser 200.

Yohimbine antagonism of the vasodepression elicited by organophosphates applied on ventral medulla oblongata

The rostral ventral surface of medulla oblongata (RVMO) has been shown to constitute a selective target for organophosphate (op) cholinesterase inhibitors. The action of soman (S) as compared with (7-nitrobenz-2-oxa-1,3 diazole)aminopentyl methylphosphonofluoridate (NBD-AP-MPF), a fluorescent organophosphate has now been examined in anesthetized cats pretreated with atropine sulphate. Blood pressure (BP), electrocardiogram (ECG) and respiration (R) were recorded. In some animals a cannula was implanted into the right lateral ventricle. Chemicals were bilaterally applied on RVMO by means of a perspex cannula and removed after 5 min. The application of 2.5 micrograms S or 60 micrograms NBD-AP-MPF elicited severe fall of BP which recovered only after 2 h in the case of the former and up to 45 min in the latter. Smaller doses produced corresponding responses of lesser magnitude. Accompanying R changes consisted in most cases of increased rate and reduced amplitude whereas in others the opposite or mixed alterations occurred. Frequently, sigh-like movements intermingled at periodic intervals with regular R deflections. The sighs were interpreted as aiming to correct blood gases balance. After application of atropine on RVMO--but not by systemic administration--BP and R were restored whereas single repeated i.v. injection of 1 microgram/kg noradrenaline produced only transient reversals without influencing the course of long lasting vasodepression. In contrast, the intraventricular administration of 250-500 micrograms yohimbine considerably reduced both the magnitude and extent of the vasodepression elicited by topically applied organophosphates. It is postulated that central alpha 2-adrenoceptors in contrast to vascular sites are likely involved in the op-induced vasodepression. The present work provides an indication that effective antagonists might be developed considering blockade of these receptors.