This chapter describes the activity, specificity and structural chemistry of Leucyl aminopeptidase (LAP). Pig kidney LAP is maximally active between pH 9 and 9.5, and cleaves a variety of amino acid amides, dipeptides and other compounds. All tested substances with an N-terminal l-amino acid are hydrolyzed and compounds that have l-leucyl residues in the N-terminal position are the preferred substrates. Peptides having proline in PI are not cleaved by LAP. Esters are also substrates of the enzyme, although they are cleaved at about 10% of the rate of the corresponding amides. A comparison of the activities of cattle lens and pig kidney LAP on amino acid amides, aminoacyl-β-naphthylamides and aminoacyl-p-nitroanilides showed that the general specificities of the two enzymes are very similar. Cattle lens LAP is a homohexamer of 324 kDa molecular mass. Its primary structure was determined by chemical sequencing and for cattle kidney LAP from the cDNA sequence. The monomer has a mixed a + β structure and consists of an N-terminal domain and a catalytic C-terminal domain. The six active sites are located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 Å and thickness 10 Å.

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein.Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, l-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity.In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

Important discoveries arise out of the confluence of various individual, historical and scientific factors. When these factors come together in the life and work of an individual of genius such as Linus Pauling, the discoveries may open up new fields for exploration by breaking down boun daries be tween indepen dent disci plines. Two examples of Pauling's pathbreaking work at the frontier of physics and chemistry are his early research in 1922 with Roscoe Dick in son in X- ray diffraction methods for determining the structures of min erals, and his European studies in 1926 in the new wave mech an ics. The first led to the five rules for stability of complex ionic crystals [[1]Pauling L. The coordination theory of the structure of ionic crystals.Festschrift zum 60. Geburstag Arnold Sommerfelds. 1928; 53: 1367-1400Google Scholar], and the second to the remarkable paper on chemical bonds, directed orbitals, stereochemistry, and magnetic properties of transition metal complexes [[2]Pauling L. The nature of the chemical bond. Application of results obtained from the quantum mechanics and from a theory of paramagnetic susceptibility to the structures of molecules.J. Am. Chem. Soc. 1931; 53: 1367-1400Crossref Scopus (793) Google Scholar]. On the border between chemistry and biology, the first of many contributions to the function of hemoglobin appeared in 1935 [3Pauling L. The oxygen equilibrium of hemoglobin and its structural interpretation.Science. 1935; 81: 421Google Scholar, 4Pauling L. The oxygen equilibrium of hemoglobin and its structural interpretation.Proc. Natl. Acad. Sci. USA. 1935; 21: 186-191Crossref PubMed Google Scholar] and in the following year Pauling and Charles Coryell showed that the uptake of oxygen changed the magnetic state of the iron from paramagnetic to diamagnetic, thus indi cating a direct interaction between oxygen and the ferrous ion. Working as he did at the edge of known science, Pauling ran the risk of occasionally being wrong, but even then his work pointed to fertile areas for future research. In the period 1936– 1939 conversations with Karl Landsteiner led Pauling to carry out many subsequent studies of hapten– antibody reactions (with Dan H Campbell and David Pressman). However, Pauling's proposal for the process of folding of antibodies so that they became complementary to antigens [[5]Pauling L. A theory of the structure and the process of formation of antibodies.J. Am. Chem. Soc. 1940; 62: 2643-2657Crossref Scopus (498) Google Scholar] was later shown to be a more complex process which involved both molecular recognition and genetic selection. Nevertheless, the basic idea of molecular complementarity mediated by the weak forces such as hydrogen bonds, salt links, dipolar forces and van der Waals forces in biological systems became a central theme of later structural investigations by Pauling and by others whom he influenced. It was my privilege to be at Caltech during 1941– 1946 and to hear Pauling's lectures and to be directed in research by him as well as by Edward W Hughes, Verner Schomaker, James H Sturdi vant and Robert B Corey. His lectures were per formances: an easy first third, a masterful second third and a challenging final third — in short, a model of inspiring teaching. Occasionally, he would sit or lie on the laboratory bench to “improve the circulation in his brain”, and sometimes in the quantum mechanics course he would arrive carefully prepared with a ‘spontaneous’ answer to an anticipated question. Most of us attended the lectures on chem i cal bonding year after year, since new material was continually being presen ted. From Pauling, I learned how to choose in teresting and significant re search problems, how to build a solid background of well-established facts, and how to make intuitive leaps of conjecture [[6]Pauling L. The genesis of ideas.in: Rinkel M Specific and Non-specific Factors in Psychopharmacology (Proceedings of the Third World Congress of Psychiatry. Philosophical Library, New York1963: 44-47Google Scholar] which were then subjected to in tense scrutiny. An occasional mistake, even when published, was not as bad as lowering one's sights to less challenging research. In the spring of 1946, near the end of my stay at Caltech, Pauling suggested that I use the then new electron microscope at Berkeley to obtain images of hapten– antibody complexes. Although the resolution was not sufficient for a successful result, the experiment demonstrated Pauling's early recognition of the potential of the electron microscope for structural studies in biochemistry. In 1932, Pauling deduced the planarity of amide groups, and this was later supported by Robert Corey's structure of diketopiperazine [[7]R.B. Corey, The crystal structure of diketopiperazine, J. Am. Chem. Sci. USA 60 1598-1604.Google Scholar]. In 1948, Pauling was ill with influenza in Oxford when he drew a polypeptide chain on paper and folded it at the Cα positions to make a helical structure containing planar peptide linkages and having 3.6 amino acid residues per turn. A major departure from all previous proposals was the conclusion that there need not be an integral number of amino acids per turn of helix. Following a preliminary account in 1950 [[8]Pauling L. Corey R.B. Two hydrogen-bonded spiral configurations of the polypeptide chain.J. Am. Chem. Soc. 1950; 72: 5349Crossref Scopus (117) Google Scholar] and a longer paper in 1951 [[9]Pauling L. Corey R.B. Branson H.R. Two hydrogen-bonded helical configurations of the polypeptide chain.Proc. Natl. Acad. Sci. USA. 1951; 37: 205-210Crossref PubMed Scopus (1955) Google Scholar], there appeared a series of seven papers, including the β-sheet structures, which transformed (and greatly increased!) the contents of the Proceedings of the National Academy of Sciences relating to biochemistry and molecular biology. Immediate experimental support for the α-helical structure was found by Max Perutz in the 1.5 å spacing of oriented fibrous proteins. Later detailed studies of protein structures showed them to have α-helical contents averaging about one-third of the organized structure, although the content varies from zero to about 85% among different globular protein structures. Pauling also contributed to transition state theory. In 1930, JBS Haldane [[10]Haldane J.B.S. Enzymes. Longmans Green, London1930: 182Google Scholar] amended Emil Fischer's lock (enzyme) and key (substrate) model to say, “using Fischer's lock and key simile, the key does not fit the lock perfectly, but exercises a certain strain on it.” In 1948, Pauling made a more precise statement which incorporated the beginning of the chemical transformation along the reaction pathway [[11]Pauling L. The nature of forces between large molecules of biological interest.Nature. 1948; 161: 707-709Crossref PubMed Scopus (453) Google Scholar]: ‘I think that enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyze.” This formulation became a significant stimulus to the study of enzyme mechanisms and the design of organic molecules which inhibit specific enzymes as a ‘rational’ method of drug design. Another major contribution was to our understanding of the origins of molecular disease. Pauling became aware of the sickle-cell anemia problem in 1945 during a lecture by William Castle. This disease is caused by aggregation of abnormal hemoglobin molecules within the erythrocyte at low oxygen pressures. Pauling and his coworkers [[12]Pauling L. Itano H.A. Singer S.J. Wells I.C. Sickle cell anemia, a molecular disease.Science. 1949; 109: 443PubMed Google Scholar] showed that the hemoglobins from normal and sickle cells had different mobilities and suggested that there were differences between the numbers of acidic or basic groups within the hemoglobin molecule. These differences were thought to influence the aggregation of the abnormal hemoglobin. In this study they identified for the first time a molecular disease. Soon thereafter, Vernon Ingram [[13]Ingram V.M. A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin.Nature. 1956; 178: 792-794Crossref PubMed Scopus (472) Google Scholar] showed that Glu6 in the β-chain of hemoglobin was replaced by valine. In a later study of mutations [[14]Zuckerkandl E. Pauling L. Molecular disease, evolution and genic heterogenetity. In.in: Kasha M. & Pullman B Horizons in Biochemistry (Szent-Györgyi Dedicatory Volume. Academic Press, New York1962: 189-225Google Scholar], Emil Zuckerkandl and Pauling related the diverse fields of molecular biology, genetics and paleontology by the proposal that changes in amino acid sequences in hemoglobin were associated with the times of divergence in evolution. A fitting tribute to Pauling ”s achievements is given in the words of Francis Crick [[15]Crick F. The impact of Linus Pauling on molecular biology: a reminiscence.in: Zewail A The Chemical Bond. Structure and Dynamics. Academic Press, New York1992: 87-98Google Scholar]: “It was because of Linus Pauling that our approach to the structural side of things had the character that it had, and it was successful because he had the right set of ideas. So we should salute Linus Pauling not only for the wonderful things that he has done for chemistry and in particular his realizing the importance of quantum mechanics for chemistry and applying it as a chemist (as opposed to just a theorist) — but also for his absolute seminal role in getting molecular biology started.” .Figure 2Linus Pauling in 1985. Photographs courtesy of William Lipscomb.View Large Image Figure ViewerDownload Hi-res image Download (PPT) William N Lipscomb, Department of Chemistry, Gibbs Chemical Laboratories, Harvard University, Cambridge, MA 02138, USA.

AbstractDie ungewöhnliche Bindung von Bor in Organoboranen oder Oligoborclustern tritt nicht nur in diamagnetischen Molekülen auf, sondern auch in paramagnetischen Systemen, die gemischtvalente Verbindungen und Oligoboran/Carboran‐Clusterradikale umfassen. Das Bild zeigt das einfach besetzte Molekülorbital des Radikalions [C4B8R4H8].− gemäß DFT‐Rechnungen.magnified imageParamagnetische Verbindungen mit mindestens teilweise borzentriertem Elektronenspin lassen sich herstellen, indem entweder durch planare, π‐konjugierte organische Systeme verbrückte Boratome als idealtypische Elektronenakzeptoren verwendet werden oder indem die dreidimensionale, delokalisierte Bindung in mehrkernigen Boranen, Halogenboranen oder Carboranclustern genutzt wird. Das Konzept der Gemischtvalenz kann so von organischen und Übergangsmetallverbindungen auf Verbindungen der Hauptgruppenelemente übertragen werden. Mithilfe der Dichtefunktionaltheorie lässt sich die sehr variable Spinverteilung nachvollziehen.

AMP binding sites are commonly used by nature for allosteric regulation of enzymes controlling the production and metabolism of carbohydrates and lipids. Since many of these enzymes represent potential drug targets for metabolic diseases, efforts were initiated to discover AMP mimics that bind to AMP-binding sites with high affinity and high enzyme specificity. Herein we report the structure-guided design of potent fructose 1,6-bisphosphatase (FBPase) inhibitors that interact with the AMP binding site on FBPase despite their structural dissimilarity to AMP. Molecular modeling, free-energy perturbation calculations, X-ray crystallography, and enzyme kinetic data guided our redesign of AMP, which began by replacing the 5'-phosphate with a phosphonic acid attached to C8 of the adenine base via a 3-atom spacer. Additional binding affinity was gained by replacing the ribose with an alkyl group that formed van der Waals interactions with a hydrophobic region within the AMP binding site and by replacing the purine nitrogens N1 and N3 with carbons to minimize desolvation energy expenditures. The resulting benzimidazole phosphonic acid, 16, inhibited human FBPase (IC50 = 90 nM) 11-fold more potently than AMP and exhibited high specificity for the AMP binding site on FBPase. 16 also inhibited FBPase in primary rat hepatocytes and correspondingly resulted in concentration-dependent inhibition of the gluconeogenesis pathway. Accordingly, these results suggest that the AMP site of FBPase may represent a potential drug target for reducing the excessive glucose produced by the gluconeogenesis pathway in patients with type 2 diabetes.

Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.

Excessive glucose production by the liver coupled with decreased glucose uptake and metabolism by muscle, fat, and liver results in chronically elevated blood glucose levels in patients with type 2 diabetes. Efforts to treat diabetes by reducing glucose production have largely focused on the gluconeogenesis pathway and rate-limiting enzymes within this pathway such as fructose-1,6-bisphosphatase (FBPase). The first potent FBPase inhibitors were identified using a structure-guided drug design strategy (Erion, M. D.; et al. J. Am. Chem. Soc. 2007, 129, 15480-15490) but proved difficult to deliver orally. Herein, we report the synthesis and characterization of a series of orally bioavailable FBPase inhibitors identified following the combined discoveries of a low molecular weight inhibitor series with increased potency and a phosphonate prodrug class suitable for their oral delivery. The lead inhibitor, 10A, was designed with the aid of X-ray crystallography and molecular modeling to bind to the allosteric AMP binding site of FBPase. High potency (IC50 = 16 nM) and FBPase specificity were achieved by linking a 2-aminothiazole with a phosphonic acid. Free-energy perturbation calculations provided insight into the factors that contributed to the high binding affinity. 10A and standard phosphonate prodrugs of 10A exhibited poor oral bioavailability (0.2-11%). Improved oral bioavailability (22-47%) was achieved using phosphonate diamides that convert to the corresponding phosphonic acid by sequential action of an esterase and a phosphoramidase. Oral administration of the lead prodrug, MB06322 (30, CS-917), to Zucker Diabetic Fatty rats led to close-dependent inhibition of gluconeogenesis and endogenous glucose production and consequently to significant blood glucose reduction.

Excessive glucose production by the liver coupled with decreased glucose uptake and metabolism by muscle, fat, and liver results in chronically elevated blood glucose levels in patients with type 2 diabetes. Efforts to treat diabetes by reducing glucose production have largely focused on the gluconeogenesis pathway and rate-limiting enzymes within this pathway such as fructose-1,6-bisphosphatase (FBPase). The first potent FBPase inhibitors were identified using a structure-guided drug design strategy (Erion, M. D.; et al. J. Am. Chem. Soc. 2007, 129, 15480-15490) but proved difficult to deliver orally. Herein, we report the synthesis and characterization of a series of orally bioavailable FBPase inhibitors identified following the combined discoveries of a low molecular weight inhibitor series with increased potency and a phosphonate prodrug class suitable for their oral delivery. The lead inhibitor, 10A, was designed with the aid of X-ray crystallography and molecular modeling to bind to the allosteric AMP binding site of FBPase. High potency (IC50 = 16 nM) and FBPase specificity were achieved by linking a 2-aminothiazole with a phosphonic acid. Free-energy perturbation calculations provided insight into the factors that contributed to the high binding affinity. 10A and standard phosphonate prodrugs of 10A exhibited poor oral bioavailability (0.2-11%). Improved oral bioavailability (22-47%) was achieved using phosphonate diamides that convert to the corresponding phosphonic acid by sequential action of an esterase and a phosphoramidase. Oral administration of the lead prodrug, MB06322 (30, CS-917), to Zucker Diabetic Fatty rats led to dose-dependent inhibition of gluconeogenesis and endogenous glucose production and consequently to significant blood glucose reduction.

Motivated by the recent discovery of unusual "hydrogen bonding"-like interaction between a borane system and benzene molecules in a molecular crystal, we carried out quantum mechanical calculations on a model complex, diborane-benzene cluster. The aim is to understand the nature of this unique interaction, which is expected to play an essential role in this novel class of molecular crystals. As analyzed in the present study, the interaction between diborane and benzene is special in the following aspects: (1) this interaction is mostly dispersive; (2) the observed pseudodirectionality with one of the diborane bridge hydrogen directed toward the benzene centroid minimizes the van der Waals contact; and (3) in the "hydrogen bond" map, this interaction is located in a unique region, which is presently populated by a few known molecular complexes with very different chemical characteristics. It is anticipated that the results from the present analysis will provide meaningful guidance for molecular engineering with diborane-benzene as a building block and for stabilization of this and possible other hydrogen bonds by dispersive contributions.

An X-ray diffraction study to 2.0 A resolution shows that this enzyme, ATCase, is in the T-state (the c3 to c3 distance is 45.2 A) when ATCase is bound to carbamyl phosphate (CP) and to L-alanosine (an analogue of aspartate). This result strongly supports the kinetic results that alanosine did not inhibit the carbamylation of aspartate in the normal reaction of native ATCase plus CP and aspartate [Baillon, J., Tauc, P., and Hervé, G. (1985) Biochemistry 24, 7182-7187]. The structure further reveals that the phosphate of CP is 4 A away from its known position in the R-state and is in the T-state position of P(i) in a recent study of ATCase complexed with products, phosphate (P(i)) and N-carbamyl-L-aspartate [Huang, J., and Lipscomb, W. N. (2004) Biochemistry 43, 6422-6426]. Moreover, the alanosine position in this T-state is somewhat displaced from that expected for its analogue, aspartate, from the R-state position. The relations of these structural aspects to the kinetics are presented.

As the trends in integrated circuit fabrication follow Moore's Law to smaller feature sizes, one trend seen in lithographic technology is the continually increasing use of optical enhancements such as Optical Proximity Correction (OPC). Small size perturbations are designed into the nominal feature shapes on the reticle such that the intended shape is printed. Verifying the success of OPC is critical to ramp-up and production of new process technologies. CD-SEMs are imaging tools which are capable of measuring feature sizes in any part of a chip, either in a test structure or within a circuit. A new trend in CD-SEM utilization is the implementation of automated recipe generation of complex CD-SEM recipes. The DesignGauge system uses design-to-SEM recipe creation and data collection. Once the recipe creation flow is implemented, the task of recipe creation can be accomplished within minutes. These applications enable a CD-SEM to be utilized to collect data for very complex OPC CD-SEM recipe runs which measure many different unique linewidths, spaces, and pattern placements within a circuit to check OPC success and lithographic fidelity. The data collection can provide accurate data results that can be utilized for comparing achieved feature measurements to nominal values from the design layout. This new application adds much value to the CD-SEM compared to other technologies such as OCD, as it completes the evaluation of in-circuit behavior to test structures in a scribe lane, something OCD currently cannot do. The present work evaluates the capabilities of DesignGauge, which is available for the latest-generation Hitachi S-9380II CD-SEMs. The evaluation includes rigorous tests of navigation, pattern recognition success rates, SEM image placement, throughput of recipe creation and recipe execution.

The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy- d - arabino -heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coli encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the ARO4 promoter drives E. coli aroH . The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy- d - arabino -heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced.

In type 2 diabetes, the liver produces excessive amounts of glucose through the gluconeogenesis (GNG) pathway and consequently is partly responsible for the elevated glucose levels characteristic of the disease. In an effort to find safe and efficacious GNG inhibitors, we targeted the AMP binding site of fructose 1,6-bisphosphatase (FBPase). The hydrophilic nature of AMP binding sites and their widespread use for allosteric regulation of enzymes in metabolic pathways has historically made discovery of AMP mimetics suitable for drug development difficult. By using a structure-based drug design strategy, we discovered a series of compounds that mimic AMP but bear little structural resemblance. The lead compound, MB05032, exhibited high potency and specificity for human FBPase. Oral delivery of MB05032 was achieved by using the bisamidate prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of an esterase and a phosphoramidase. MB06322 inhibited glucose production from a variety of GNG substrates in rat hepatocytes and from bicarbonate in male Zucker diabetic fatty rats. Analysis of liver GNG pathway intermediates confirmed FBPase as the site of action. Oral administration of MB06322 to Zucker diabetic fatty rats led to a dose-dependent decrease in plasma glucose levels independent of insulin levels and nutritional status. Glucose lowering occurred without signs of hypoglycemia or significant elevations in plasma lactate or triglyceride levels. The findings suggest that potent and specific FBPase inhibitors represent a drug class with potential to treat type 2 diabetes through inhibition of GNG.

A series of full- and half-sandwich metallacarboranes were synthesized from the reaction of MCl2 (M = Co, Fe) and closo-exo-y,x-Li(L)-1-Li(L)-2,n-(SiMe3)(2)-2,n-C2B4H4 (Y = 4, x = 5, L = 2 THF, n = 3 (1); y = 4, x = 5, L = TMEDA, n = 3 (2); y = 5, x = 6, L = 2 THF, n = 4 (3); y = 5, x = 6, L = TMEDA, n = 4 (4)) in 1: 1 molar ratios in benzene. The metallacarborane complexes exo-4,4',5,5'-Fe(TMEDA)-commo-1,1'-Fe[2,3-(SiMe3)(2)-2,3-C2B4H4](2) (5), M+[commo-1,1'-Co{2,3-(SiMe3)(2)-(2,3-C2B4H4)}(2)]-(M = CoCl (6a), CO3(TMEDA)(3)Cl-5 (6b)), commo-1,1'-M[2,4-(SiMe3)(2)-2,4-C2B4H4](2) (M = FeH (7), CoH (8)), and 1-(TMEDA)-closo-2,4-(SiMe3)(2)-1,2,4-MC2B4H4 (M = Co (9)) were isolated in yields ranging from 80 to 92%. The reaction of commo-1,1'-Ni[2,4-(SiMe3)(2)-2,4-C2B4H4](2) (10) with carefully dried TMEDA produced the charge-compensated commo-1,1'-Ni-III-[(2,4-(SiMe3)(2)-5,6-B-2-{sigma-N(Me)(CH2)(2)N(Me)(2)}-2,4-C2B2H2)(2',4'-(SiMe3)(2)-2',4'-C2B4H4)] (12) and 1,2-(SiMe3)(2)-closo-1,2-C2B4H4. With the exception of 5 and 12, all of the commo-metallacarboranes involved metal oxidation in addition to ligation, with M-0 as a coproduct. All compounds were characterized by analysis and infrared spectroscopy, and 5, 6b, 7, 9, and 12 were also characterized by X-ray diffraction analysis. Because of the paramagnetic nature of either the products or their counterions, only 8 could be characterized by H-1, B-11, and C-13 NMR spectroscopy; its H-1 NMR spectrum showed a broad resonance at delta - 18.9 attributable to the Co-bound H. The paramagnetic compounds were characterized by EPR spectroscopy. The electrochemical reduction of 10 produced a Ni(III) metallacarborane that was characterized by EPR spectroscopy and found to be a 3d(7) species related to nickelocenium derivatives. All results are consistent with a mechanism involving the initial formation of a half-sandwich metallacarborane, which can be isolated or can undergo a disproportionation process to give the full-sandwich products.

Structural changes during the R-to-T transition of fructose-1,6-bisphosphatase (EC 3.1.3.11) form a hierarchy, in which structural changes at one level are supported by those at the other levels. The quaternary conformational changes involve a 17° rotation between the upper and lower dimers, and a 3.4° rotation between monomers in a dimer. Within monomers, the FBP domain, which remains rigid during the R-to-T transition, rotates 2.30 relative to the AMP domain, which undergoes significant structural reorientations. The most important of these reorientations are the newly identified partially ordered loop residues 55-61 in the T state and reorientations of helices Hi, H2, and H3. Supporting these structural changes are numerous readjustments of hydrogen bonding and van der Waals interactions throughout the entire tetrameric protein. Propagation of structural changes during the R-to-T transition relies primarily on helices Hi, H2, H3, and loop 50-72. The change that begins at the AMP site causes reorientation of Hi, H2, and H3 and changes of interactions across the Cl-C4 (C2-C3) interface. These changes may propagate down Hi, H2, H3, and loop 50-72 to affect interactions across the Cl-C2 (C3-C4) and Ci-C3 (C2-C4) interfaces. AMP inhibition is most probably caused by reduced metal binding affinity due to structural changes of metal ligands (Glu97, Asp'l8, and Asp'21) in the active site. The eight-stranded fl-sheet, particularly the ,8-strand B3, which connects Lys' 2 and Tyr"13 of the AMP site with Aspll8 and Asp12' of the metal site, may be responsible for communication between the AMP and active sites. Additional structural changes that support such communication include reorientation of the FBP domain and Hi, H2, and H3 relative to the eight-stranded fl-sheet, and new conformations of loop 54-72 in the T state as AMP binds. Fructose-1,6-bisphosphatase (Fru-1,6-Pase, EC 3.1.3.11) is a key regulatory enzyme in the gluconeogenic pathway (1-5). It converts D-fructose 1,6-bisphosphate (Fru-1,6-P2) to D-fructose 6-phosphate (Fru-6-P) and phosphate. A divalent metal ion such as Mg2+, Mn2+, Zn2+, or Co2+ is required for catalytic activity (1, 6), except for the brain enzyme (7). Early purification of the rabbit liver enzyme yielded two forms of enzyme due to proteolytic cleavage. The cleaved enzyme has maximum activity at pH 8.4-9.4 and is designated "alkaline enzyme." The intact natural enzyme, on the other hand, has maximal activity at pH 7.6 and is designated "neutral enzyme." Cleavage in the alkaline enzyme is now identified to occur at the loop residues 54-67. Coordinated regulation (2-5, 8) of mammalian Fru-1,6Pase occurs by AMP at an allosteric site and by D-fructose 2,6-bisphosphate (Fru-2,6-P2) at the active site and probably also at an allosteric site. These regulatory mechanisms minimize, especially at low Fru-2,6-P2 concentration, a loss of ATP in a futile cycle catalyzed by Fru-1,6-Pase in gluconeogenesis and the opposing enzyme, phosphofructokinase, in x x FIG. 1. Schematic of R (Left) and T (Right) states of Fru-1,6Pase, where monomers are labeled clockwise as Cl, C2, C3, and C4. The x, y, and z axes correspond to three molecular twofold axes, and the origin (0) to the center of each tetrameric enzyme. glycolysis. For the yeast enzyme, phosphorylation may play a role in regulation (9). Kinetics studies of mammalian Fru-1,6-Pases have indicated that AMP binds noncompetitively and slightly cooperatively to the enzyme's allosteric site (2, 4, 5, 10). The Ki values for inhibition by AMP vary: <1 ,M for the rabbit skeletal enzyme (11), 10-20 AM for the liver and kidney enzymes (12), 80-200 ,uM for the yeast enzymes (13, 14), and no inhibition for the chloroplast and bumblebee flight muscle enzymes (15). Sequence conservation of AMP binding residues is poor, except for mammalian enzymes. Metal ions, inhibitors, and effectors show interactive effects. Interactions are seen between Mg2+ and Zn2+ in the rabbit muscle enzyme, and synergistic interaction occurs between inhibition by AMP and by inhibition by Fru-2,6-P2 (3, 8, 16-20). A third example is the inhibition of metal binding at the catalytic metal sites by an analog ofAMP (21), whereas catalytic metal binding is enhanced by binding of substrate or products (17). For substrate binding, homotropic cooperativity is small or absent for mammalian Fru-1,6-Pase, although it is large for the spinach enzyme. Negative cooperativity occurs in the binding ofthe inhibitor Fru-2,6-P2 (22). Crystallographic studies of pig kidney Fru-1,6-Pase and its complexes yielded two quaternary conformations, the R and the T forms, which differ by a 170 rotation of the lower, C3C4 dimer relative to the upper, C1C2 dimer (23, 24) (Fig. 1). The T-form structures include the Fru-6-P/AMP/Mg complex (2.5-A resolution) (23) and the AMP complex (2.5-A resolution) (24).* The R-form structures include Fru-1,6-Pase complexed with Fru-6-P (2.1-A resolution) (25), Fru-2,6-P2 (2.6-A resolution) (26), Fru-1,6-P2 (2.5-A resolution) (27), 2,5anhydroglucitol 1,6-bisphosphate (2.6to 3.0-A resolution) (27), and 2,5-anhydromannitol 1,6-bisphosphate (2.6to Abbreviations: Fru-1,6-Pase, fructose-1,6-bisphosphatase; Fru-1,6P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fructose 2,6-bisphosphate; Fru-6-P, fructose 6-phosphate. *The early report of AMP binding to the AMP sites of the R-form enzyme was an artifact of incomplete refinement. Further refinement shows no AMP in the AMP sites of the R-form enzyme. 2132 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 90 (1993) 2133 FIG. 2. Ribbon drawing showing secondary structures and loops of the Cl monomer. H, helix; B, (-strand; L, loop; T, turn. Residue ranges: N-ter, 9-12; Hi, 12-24; Li, 25-27; H2, 28-50; LOOP, 50-72; H3, 72-88; L2, 89-91; Bi, 92-95; Ti, 96-99; L3, 100-102; B2, 103-106; L4, 107-112; B3, 113-118; L5, 119-122; H4, 123-127; L6, 128-131; B4, 132-139; L7, 140-148; H5, 149-153; L8, 154-159; B5, 160-167; T2, 168-170; B6, 171-176; T3, 177-180; B7, 181-187; T4, 188-191; B8, 192-199; L9, 200-206; B9, 207-210; L10, 211-220; H6, 221-232; Lll, 233-239; B10, 240-243; L12, 244-246; H7, 247-258; Bli, 260-264; L13, 265-275; H8, 276-291; L14, 292-293; B12, 294-2%; T5, 297-300; L15, 301-315; B13, 316-319; H9, 320-333. As called to our attention by Bobby Baum (personal communication), there are similarities of sequences of Fru-1,6-Pase and inositol monophosphatase (29). Both have an a/3a,8a type of structure. 3.0-A resolution) (27), in the presence or absence of metal ions. The allosteric AMP binding site is 28 A from the nearest substrate binding site. Regulation of allosteric enzymes is often achieved through quaternary conformational changes induced by substrate or effector binding. A recent structural comparison (ref. 28 and references therein) of R and T forms of hemoglobin and aspartate carbamoyltransferase from Escherichia coli (EC 2.1.3.2), glycogen phosphorylase from rabbit muscle (EC 2.4.1.1), and phosphofructokinase from Bacillus stearothermophilus and E. coli (EC 2.7.1.11) showed that the quarternary conformational change, which involves primarily rotations of subunits with very little translation, often preserves most or all of the symmetry and individual secondary structures of proteins. These structural analyses provide us with important insights into understanding possible pathways of allosteric transitions. What remain to be unraveled are descriptions at the atomic level and detailed mechanical pictures of such quatemary conformational changes.

The effector-regulated allosteric mechanism of yeast chorismate mutase (YCM) was studied by normal mode analysis and targeted molecular dynamics. The normal mode analysis shows that the conformational change between YCM in the R state and in the T state can be represented by a relatively small number of low-frequency modes. This suggests that the transition is coded in the structure and is likely to have a low energetic barrier. Quantitative comparisons (i.e. frequencies) between the low-frequency modes of YCM with and without effectors (modeled structures) reveal that the binding of Trp increases the global flexibility, whereas Tyr decreases global flexibility. The targeted molecular dynamics simulation of substrate analog release from the YCM active site suggests that a series of residues are critical for orienting and "recruiting" the substrate. The simulation led to the switching of a series of substrate-release-coupled salt-bridge partners in the ligand-binding domain; similar changes occur in the transition between YCM R-state and T-state crystal structures. Thus, the normal mode analysis and targeted molecular dynamics results provide evidence that the effectors regulate YCM activity by influencing the global flexibility. The change in flexibility is coupled to the binding of substrate to the T state and release of the product from the R state, respectively.

Structures of the R-state of Escherichia coli ATCase maintained with carbamyl phosphate and succinate, phosphonoacetamide and malonate, or N-phosphonacetyl-l-aspartate (PALA) have previously been made in the space group P321, in which the two independent r (regulatory) and two independent c (catalytic) chains are repeated by crystallographic symmetry to yield the holoenzyme c(6)r(6), ((c(3))(2)(r(2))(3)). The exploration of a new crystalline R-state P2(1)2(1)2(1) was undertaken to examine the c(3).c(3) expansion of 11 A in the T-to-R transition, and to further test whether intermolecular contacts influence the binding of PALA. The results show that the expansion along the 3-fold axis is 10 A, and that the binding modes of the six crystallographic independent PALA molecules are virtually identical to one another, and to modes described previously. As further test, the PALA, a bisubstrate analogue, was displaced by citrate and phosphate, where citrate is an analogue of product carbamylaspartate. The results support the conclusions about the binding of the three previously studied analogues, and further support, within about 0.5 A, the structure proposed for the transition state [Gouaux, J. E., Krause, K. L., and Lipscomb, W. N. (1987) Biochem. Biophys. Res. Commun. 142, 893-897; Jin, L., Stec, B., Lipscomb, W. N., and Kantrowitz, E. R. (1999) Proteins: Struct., Funct., Genet. 37, 729-742].

AbstractLeucine aminopeptidase belongs to the metallohydrolase family M17. In this family, two X‐ray diffraction structures are known for bovine lens LAP (blLAP) and for theEscherichia coliaminopeptidase A (PepA). These two peptidases are large homohexamers of 32 symmetry, and both have a large central solvent cavity near the active sites. Each active center contains two Zn2+separated by 3.0 Å. In PepA, maximal activity occurs in the presence of Mn2+. blLAP is activated by Mn2+, Mg2+or Co2+, but zinc is bound most strongly and is supposed to be the physiological cofactor. In the proposed catalytic mechanism, both metal ions stabilize substrate binding and formation of the transition state. In addition, a lysine side chain contributes by polarizing the substrate's carbonyl group, and an arginine side chain binds a bicarbonate anion (or carbonate ion) that is in a position to act, at least in PepA, as a general base to deprotonate the zinc‐bound water nucleophile.

The structure of aspartate transcarbamylase of Escherichia coli ligated to products (phosphate and N-carbamyl-L-aspartate) has been determined at 2.37 Angstrom resolution (R-factor = 0.23, R-free = 0.27). Results might indicate a product release mode, rather than close analogues to the transition state like those found in our earlier studies of other ligands (N-phosphonacetyl-L-aspartate, carbamyl phosphate plus malonate, phosphonoacetamide plus malonate, or citrate plus phosphate). Ordered product release, first carbamylaspartate (CLA) and then phosphate, might be facilitated by a 4 Angstrom movement of phosphate from the substrate-analogue position to the product (phosphate) binding position, and by a somewhat similar release movement of the other product (CLA) relative to its analogue (citrate). This movement is consistent with earlier studies of binding of either pyrophosphate or phosphate alone.