FEBRUARY 1971
`
`VOLUME 68
`
`NUMBER 2
`
`PROCEEDINGS OF THE
`
`National
`Academy of
`Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2137 - 1/7
`
`

`
`THE PROCEEDINGS OF THE
`National
`Academy of
`Sciences
`OF THE UNITED STATES OF AMERICA
`
`O.fficers
`of the
`Academy
`
`Editorial Board
`of the
`Proceedings
`
`PHILIP HANDLER President
`G. B . KrsTIAKOWSKY Vice President
`MERLE A . TuvE Home Secretary
`HARRISON BROWN Foreign Secretary
`E . R. ProRE Treasurer
`
`Jmm T. EDSALL Chairman
`WAL'l'ER KAUZMAKN Vice Chairman
`JAMES V. NEEL Vice Chairman
`MERLE A . TuvE Home Secretary
`HARRISON BROWN Foreign Secretary
`E. R. ProRE Treasurer
`c. B. ANFINSEN
`FRANK BRINK, JR.
`BERNARD D . DAVIS
`KINGSLEY DAVIS
`Lours B . FLEXNER
`WILLIAM A. FOWLER
`LEo GoLDBERG
`RoBERT A. Goon
`
`lVIARK KAC
`GoRDON J. F. MAcDoNALD
`ELLIOTT W . lVIoNTROLL
`ARTHUR B. PARDEE
`THEODORE T. PucK
`RrcHARD B. RoBERTS
`PAUL A. SAMUELSON
`EMIL L . SMITH
`
`Managing Editor: F. PETER WooDFORD
`Associate Managing Editor:
`JEROLD A. LAST
`
`'Editorial correspondence: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,
`Avenue, Washington, D.C. 20418.
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`
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`-4»:..<=u.s.g=-rs-s-n.—,:x-pus:-an-.-new-wf
`
`Contents (Continued from outside back cover)
`
`BOTANY
`
`G lCNlD’1‘ICS
`
`IMMUNOLOGY
`
`MEDICAL
`SCIENCES
`
`MIcRonIoLoGY
`
`PATHOLOGY
`
`PHYSIOLOGY
`
`CORRECTIONS
`
`.
`.
`.
`.
`Loss of feedback control of hydroxymethylglutaryl—coenzyme A reductase in hepatomas .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Marvin D. Siperstein, Ann M. Gyde, and Harold P. Morris
`Association of chromosomal RNA with repetitive DNA
`Yuriy Ill. Sivolap and James Bonner
`Characterization of the initial peptide of Qfl RNA polymerase and control of its synthesis .
`.
`.
`.
`.
`.
`.
`.
`.
`.
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`.
`.
`.
`.
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`.
`.
`.
`.
`. Lawrence Skogerson, Donald Roafa, and Philip Leder
`DNA—dependent RNA polymerase from yeast mitochondria
`.
`.
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`.
`-
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`.
`.
`.
`. Ming-Jer Tsai, Georg Michaelis, and R. S. C'riddle
`The comparative specificity of acid proteinases
`. Irene M. Voynick and Joseph S. F7‘ut0n
`
`-
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Cytokinin and growth of excised roots of Bryophgllum calgcinimn .
`.
`.
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`.
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`.
`.
`.
`.
`.
`.
`.
`William. J. Robbins and Annette Hervey
`Cyunophycin granules from the blue-green alga Anabaena cylindrica: a reserve material consisting of co-
`polymers of aspartic acid and arginine .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Robert D. Simon
`Zoospore germination in Blastocladiella ernersonir? cell differentiation without protein synthesis? .
`.
`.
`...................
`.D.R.SollandD.R.Sonneborn
`Revised identification of the chromophore of a cell division factor from crown gall tumor cells of Vinca
`rosca L. (Correction) .
`. Henry N. Wood
`
`Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. B. G. Braclcett, W. Baranska, W. Sawiclci, and H. Koprowski
`Temperature-dependent surface changes in cells infected or transformed by a. thermosensitive mutant of
`polyoma virus .
`.
`.
`.
`.
`.
`.
`.
`. Walter Eclchart, Renato Dulbecco, and Illax M. Burger
`DNA-induced transformation in Drosophila: genetic analysis of transformed stocks .
`.
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`.
`.
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`.
`.
`.
`. Allen S. Fox, Sci Bgung Yoon, and William M. Gelbart
`Immunochemical specificity of lactate dehydrogenase—X.
`.
`Erwin G0lllb9?‘0
`.
`.
`Spontaneous recombination in Drosophila mclanogaster males
`.
`Yaichiro Hiraizumi
`.
`Characterization of mitochondrial ribosomes from yeast
`.
`. H. Morimoto and H. 0. Halvorson
`.
`.
`On cell lethals in Drosophila
`.
`.
`.
`.
`.
`.
`. Curt Stern and Chiyolco Tolcunaga
`Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties Of 21
`77W-7+/77l€¢-7" diploid .
`Ching-Hsiang Sn and Ronald C. Greene
`
`Membrane-associated immunoglobulin of rabbit peripheral blood lymphocytes: allelic exclusion at the b
`locus
`.
`.
`.
`.
`.
`. Joseph M. Davie, William E. Paul, Rose G. Mage, and Margaret B. Goldman
`Enhanced immunogenicity of chemically-coated syngeneic tumor cells .
`.
`. William J. Martin, John R. Wunderlich, Freernan-Fletcher, 4...; m..’. K: Inman
`
`.
`Induced degradation of glycosaminoglycans in Hurler’s and Hunter's syndromes by plasma infusion.
`.........................NicolaD~iFerrante,
`Buford L. Nichols, Patricia V.‘ Dtmnelly, Giovanni Neri, Ruzica Hrgoocic, and Robert K. Bergland
`Presynaptic neuromuscular inhibition by porphobilinogen and porphobilin .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Daniel S. Feldman, Richard D. Levere, James S. Lieberman, Ruth A. Cardinal, and C. J. Watson
`Transformation of rat and hamster embryo cells by extracts of city smog .
`.
`.
`. Aaron E. Freeman,
`Paul J. Price, Robert J. Bryan, Robert J. Gordon, Raymond V. Gildcn, Gary J. Kcllofi, and Robert J. Huebner
`
`-
`-
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`Germination and growth of Neurospora at low water activities .
`.
`.
`.
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`.
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`.
`.
`.
`.
`.
`.
`. Gisela Wohlrab Charlang and N. H. Horowitz-
`Temperature-sensit.ive mutants of bioluminescent bacteria .
`. Thomas Cline and J. W. Hastings
`A role of aminoacyl-.tRNA in the regulation of protein breakdown in Escherichia coli
`Alfred L. Goldberg
`Induction of Staphylococcus aureus lactose permease in the absence of glycerolipid synthesis .
`.
`.
`-
`-
`LeonardMindi¢h
`Interferon and transcription of early virus-specific RNA in cells infected with simian virus 40 .
`.
`.
`-
`. Michael N. 0:1:-man and Myron J. Levin
`. Kirsten H. Walen
`
`Nuclear involvemienti in Ipoicvirius i-nfeictioln (Correction)
`
`.
`
`315-317
`387-389
`
`276-279
`
`473-477
`257-259
`
`347-348
`
`265-267
`
`459-463
`
`509
`
`353-357
`
`283-286
`
`342-346
`349-352
`268-270
`324-328
`329-331
`
`367-371
`
`430-434
`
`469-472
`
`303-307
`
`383-386
`
`445-449
`
`260-262
`500-504
`362-366
`
`420-424
`
`299-302
`509
`
`Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentia-
`tion by dimethyl sulfoxide .
`Charlotte Friend, William Scher, J. G. Holland, and T07‘?-4 Sato
`
`378-382
`
`Calcium as a regulator of the proliferation of normal, but not of transformed, chicken fibroblasts in a plasma-
`containing medium
`. Samuel D. Balk
`.
`.
`.
`.
`.
`.
`.
`The hydration of sodium ions crossing theinerive membrane‘
`.
`Bertil Hille
`Hypothalamic alpha- and beta-adrenergic systems regulate both thirst and hunger in the rat
`.
`.
`.
`.
`_ Sarah Fryer Leibowitz
`
`271-275
`280-282
`
`332-334
`
`. Donald C’. Borg, Jack Fajer, Ronald H. Felton, and David Dolphin
`The 1r-cation radical of chlorophyll a
`Nuclear involvement in poxvirus infection .
`. Kirsten H. Walen
`Revised identification of the chromophore of a cell di-Vrisiion factor from crown gall tumor cells of Vinca
`rosea L.
`Henry N. Wood
`
`509
`509
`
`509
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`

`
`Contents
`
`Physical Sciences
`
`Vol. 68, No. 2
`
`February 1971
`
`CHEMISTRY
`
`Mechanism of action of thrombin on fibrinogen, 1. Synthesis of fibrino
`gen-like peptides, and their PTO‘
`teolysis by thrombin and trypsin.
`.
`.Rudolf H. Andrealta,
`Ronald K. H. Liam, and Harold A. Schema“
`Synthetic derivatives of polyethylenein
`iine with enzyme
`-like catalytic activity (synzymes) .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Irving M. Klotz, Garfield P. Roger, and Ioannis S. Scarpa
`.
`Nonprotein amino acids in the Murchison meteorite .
`. Keith A. KvenvoldcriI
`
`GEOPHYSICS
`
`l\/lA’1‘}I1«)MA’1‘ICS
`
`‘
`
`Arrnznn
`
`.
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`.
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`.
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`.
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`.
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`'
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`.
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`'
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`'
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`’
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`.
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`.'
`
`.
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`.
`
`.S. H. Lin and H..Evri'n9
`.
`.
`.
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`.Thomas H. Jordan and Joel N. Franklin
`
`.
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`.
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`.
`
`.
`
`.
`
`.Fra-ncis D.1|r[arnaglwm
`
`.lVlA'1‘IIEMA’1‘ICS Stochastic speculative price .
`
`Biological Sciences
`
`. Paul A. Samuelson
`
`BIOCIIEMISTRY
`
`The acyl dihydroxyacetone phosphate pathway for glycerolipid biosynthesis in mouse liver and Ehrlich
`ascites tumor cells
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Bernard W. Agranoff and Amiga K- Hal”
`l)rastic alteration of ribosomal RNA and ribosomal proteins in showdomycimresistant Escherichia coli
`’
`.
`.
`.
`.
`.
`.
`. Mirko Beljanski, Pierre Boargarel, and Monique Beljansl“
`.
`.
`.
`Rapid and complete purifi
`holinesterases of electric eel and erythrocyte by affinity chm‘
`matography
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Jonathan Dembitz Berman and Michael Y0’?m9_
`I)issociatioi1 of mammalian polyribosomes into subunits by puromycin
`. G‘llILl€I' Blobel and David Sabalinl
`The -zr-cation radical of chlorophyll a (Correction)
`.
`.V
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Donald C. Borg, Jack lmajer, ‘Ronald. H.' Feltori, and David Dolflhi"
`Chick embryo ribosome crystals:
`‘
`ing and functional activity in vilro .
`. Brcclc I31/W3
`Photoinactivation of aldolases by
`. L. 0. Davis, G. Riberean-Gayon, and B. L. Horeclcer
`n factor FII from Escherichia coli: evidence for a d-1131
`. Jerry S. Dubnofi” and Umadas Maitra
`er in the absence of an electron transfer
`. G. s. P. Groot, L. Kama, and G. Schulz
`etrahydrofolic acid: methionyl—tRNA trans-
`NA with mitochondrial DNA .
`.
`.
`- "
`.
`.
`.
`. A. Halbreich and M. Rabinowilz
`. Anne M. H0»!/‘wood
`la emcrsonii .
`-
`-
`'
`. Paul A. Horgcn and David H. Gflfiln
`of derivatives of. the side chai
`n of oxytocin: examples of cis-trans isom-
`.Viclor J. Hruby, A. I. Brewster, and J. A. Glasel
`ls by dibuliyryl adenosine cyclic 3’:5’-monophos‘
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. Abraham W. Hsie and Theodore T- Puck
`1311316 and testosterone
`gical characteristics of normal fibroblasts in sarcoma cells treated with
`1{.esto1‘ation of sever
`phate and its
`'
`adenosine~3’:
`.
`.
`.
`. George S. Johnson, .RolJert.M: Friednian, and Ira Pasta"
`.
`.
`.
`.
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`.
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`.
`.
`Absence of a natural inhibitor of the tRNA m
`ethylases from fetal and tumor tissues .
`. Sylvia J. Kerr
`The nature of the collagen synthesized by cult
`.
`ured human fibroblasts .
`.
`.
`.
`.
`.
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`-
`-
`_‘
`. Don L. Layman, Ermona B. McGoodwin, and George R. Mam"
`.
`‘.
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`Remitiation of RNA synthesis on transcription of chromatin DNA by Escherichia coli RNA polymerase '
`-
`_.
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`. Margaret E. Morris and Hannah Gould
`_-
`_
`Inhibition of serine proteases by arylboronic acids
`.
`. M. Phiilipp and M. L. Bender
`ion of mitochondrial ribosomes in the poky mutant of Neurospora crassa .
`.
`-
`_ Luci
`
`.
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`.
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`.
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`.
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`‘
`-
`-
`-
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`he reovirus genome .
`Samuel C’. Silvcrstein, Daniel H. Levin, and George A63
`acids in developing rat kidney cortex .
`.
`.
`-
`-
`_
`'
`. Stanton Segal, Claire Rea, and Inez Smith
`11 of the cystathione 7-synthase of NeurosP0"“
`. J. Selhub, M. A. Savin, W. Sahami, and M. Flavin
`cyte initiation factors M1 and M2
`. David A. Shafritz, David G. Laycock, and'W.. French Anderson
`.
`-
`(Continued on inside
`
`back
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`

`
` This material may be protected by Copyright law (Title 17 U.S. Code)
`
`l’rocm'rling.x- of the 1\’a(z'0nu/ A<:aclem_i/ of Sciences
`Vol. (3%, No. ‘.2, pp. 47-%—4S(),
`l<‘ebru:Lry 197]
`
`Inhibiliou of Serine Proleases by Arylhoronie Acids
`
`l\l.
`
`l’IllLIl’l’* ;\.\'l) ;\I. L. BENDER
`
`l)('3lHtl'tlll(3Il\ of Clieinistry, Northwestern Univensity, Evnnston. Ill. 6020}
`
`Vfmurmuz{Mimi .'\’0zienz.l)(ar 130, 1,070
`
`Arylhoronie acids were found to be strong
`/\llS'l‘R/\(I'|‘
`f:()lIl[)ctili\’c inhibitors of suhtilisin and chymotrypsin.
`I‘he binding constants are strongly pH dependent and
`lzive u Hammett-type plot with a slope of -0.885. The pH
`dependence, the Hammett plot, and nmr model-system
`studies imliente that inhibition is due to electron-pair
`donation by the active site histidine to the bound in-
`hibitor.
`
`The stiuly oi <-l1_\'1notr_vpsin has often been lueilituterl by the
`rise ol nonsiier.-ilie inliibitors. In :t(l(lltl()1l to serving‘ such direct
`|'um~tion.s us lzrlieling‘ essential groups of the protein, they are
`often used in .'su<<li se<:ondtu'y roles as in the determinzttion of
`‘~“‘7'.V'“1=llri<t 1'e:1.t-tiou rates in relnxution and dezteylution .~;tudie:s.
`Inhibitors (111.11 also be it put of rational purifiezttion schemes.
`. An inhibitor that binds .st1‘on,9,'ly but yet is easily l’CII‘10V:‘I.lJl0
`lrom the binding site by slight elnmges in pH can present ob-
`vious :ulv:mt:t;;es in zsueh protein studies. Equally {L(lVaI]t{I.-
`.‘s"<‘ous
`is the :ivnilul)i1ity 01' it simple ehemieal moiety which,
`Wlien atttnclieul to an otherwise nonspecific molecule, confers
`“"3 “l’ilit.\' T0 eoinpetitively bind to the desired protein. The
`boronie neirl ;__’,'l'()ll[), whose use was pioneered by I. V. Berezin,
`ct ul. (I, 2) is zm exmnple of such [L moiety. We think that the
`u.v:til:tbilit_\' of this grroiui will ,qreutly f;u-ilitztte the desigii of
`"‘””l>0umls thut may have other desirable. 1>ro1)ertie.s, sueh
`H5 =1 \'i.~‘ible f'llI'()l]Il)[)ll()l'(‘. or :1. spin label.
`
`(M"xio‘3)
`ASSOCIATIONKI
`
`pl] (lependenee of K1” (.\I“) for benzenebornnie
`1.
`Fm.
`ttt‘lll
`inhibition of
`.<.ublilisin. Ethyl
`;\’-neetyi-I,-tyrosin2tte(O),
`rtml methyl
`:\’-in-(-,l_\'l-I.-Iryploplizm:Lle(O), were used as sub-
`sl r:i.le.s.
`llulleins below [>11 (5 are neelule, between pll (i and H are
`pl1o.spl1:tle, uml ulmve pll 8 are :lm1Il()lll:L, enrbon:ile, and pyro-
`pln l.‘«‘])llll I e.
`
`M ETHO D S
`
`The inhibition eonstzmts for -.1. series of ri11,<__>;-sLil.>.stituted ben-
`zeneboronie zteids we1'e determined us it function of pH with
`ehromo1>horic Substrates in buffe1'e(:l solutions. The inhibition
`(-oiistznits were then extrzteted from time-course (1-‘mt (3) using_r
`equzttions given in Webb (4) for competitive inhibitons. Sub-
`strates used were ethyl N-zteetyl-L-tyrosiimte (.3), and methyl
`N-xx-:Lee.tyl-L—t1'y1)top11zmztte (6) for eliymotryiisin, and these
`plu.s phenyl
`liippurztte and 'p—nitro1)hen_vl zteetute (_7)
`for
`subtilisin. Buffers used \vere zreetztte, pliosplnite, ztmnlollltl, and
`<-.u.rbointt.e.
`
`l)u1'1'um-Gibson
`:L
`in
`Stoppe(l—fio\v studies were (lone
`stopped-flow :t])D2tI'iLtL1S. I11 this zt])})£t1‘z1l111S, equal zmiounts of at
`benzeneboronie acid solution (0~6 X 10” M) were mixed
`with a 2.54 X IO‘5 M p1'ofluvi11—3.20 X IO‘5 M a—eh_Vmo-
`trypsin solution. The :ib.s‘o1'l)zmee was I1'1€{1SUl‘C(l at 465 nm
`(ref. 8) 10 msee after mixing. The solvent used \V£LS pH 6.75
`pl1ospl‘1ate buffer, with an ionic strength of 0.2.
`l’>ruker 90-)/Ihz
`The 11mr studies were done at 38°C on :1
`instrument opemtiilg at 28.87 Mhz for the “IE nucleus. The
`solvent was zn1hyd1'ous methanol; methyl borute was used :1:
`un external stzmdzml.
`Three times 1'ee1'ystal1ize<l a-eliymotrypsin was ]>i1rel1u.se(l
`from Worthington llioelieniieztls, Freehold, N..l. Novo .subtili—
`sin was purclnised from the Enzyme Development Corp., (34
`Wall Street, N.Y.
`
`RESULTS
`
`Enzyme inhibition studies
`The inhibition eoiistzmt (late. for the various 1'ing—;sul)stitutetl
`benzeneboronie ue-ids was plotted as 1/K1 (in ;\I“1) versus
`pll. A bell-slnrped curve resulted in every case (:Lsi11Fig. 1).
`and eould be resolved to it 1/K1(_lim) and two pK values.
`The first })K, for every inhibitor, was near 7. This approxi-
`mzttes the pK of the enzyme czttztlytie eonstztnt, which re.sult.s
`lrom the imid:1zo1e:Lt the active site.
`
`The other pK varied with the inhibitor used; thus, this pl{
`must result from inhibitor ionization. It could not result from
`
`un enzyme-binding pK since subtili:sin shows no sueh pI{ in
`the binding of neutral substrates (7, 9). With one e.\'ee1r-
`tion, the pKs observed also tt])])1‘0XlIl121tC those in the litera-
`ture (10). We then plotted the value of the ;seeond pl{ versus
`log 1/K1(_lim‘)
`:n1r,lol)tz1.iner.l
`it least-squztres slope of -0.895
`with at correlzttion eoeflieient of 0.96. This indiezttes that the
`inhibitors’ affinity towards subtilisin approxiinzites their affin-
`ity tow2u'Ils :1. l1yd1'ox_vl ;_r;roup*. This is seen in Fig. 2.
`
`" .\l.
`
`l’. wns uli Nlll l’1'e:lo<'1orul
`
`l"el|ow,
`
`l‘.!(')r‘<—l‘.l'/'0.
`
`* Bnronie zreids 1)rol)ztbly ionize us Lewis iteids.
`
`478
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`

`
`Vol . 68, 1971
`
`Enzyme
`
`a-Chymotrypsin
`a -Chymotrypsin
`Subtilisin
`Subtilisin
`Subtilisin
`Subtili::;in
`Subtilisin
`
`* Ref. (10).
`t Ref. (16).
`
`_-\.rylboronic .-\cid;; as Inhibitors
`
`479
`
`TABLE l.
`
`Inhibition by boronic acids and benzyl alcohol
`
`ln hibi tor
`
`Ben;-.eneboroni c arid
`Benzyl alco hol
`Benzeneboroni c acid
`m-Nilrubenzeneboruni(" acid
`1n-Aminoben;-.eneboro ni c aeid
`p-IJromoben;-.eneboro ni c :wid
`Ben;-.yl alcohol (pH 8.4)
`
`K r
`
`l .U6 X 10 -< ?vi
`1.0 X lQ -2 l\[ t
`2 .2U + lQ -4 :\1
`1.04 X lQ - 5 :'If
`1 .32 X 10 - " :\I
`2.27 X w-' :'II
`1.1 X w - r :'11
`
`pE:r
`
`7.0
`none
`7.1
`7 . 0
`7.0
`7.:37
`
`pK,
`
`8 .42
`H.Ot
`8 . !)2
`7.2;)
`8 .66
`8. j()
`
`pK*
`
`8 .83
`none
`8.83
`7. 2:1
`
`8 66
`none
`
`To compare the inhibition con,;tants with t hose of a nalogous
`compounds without t he boronic acid group, t he 1\r of benzyl
`alcohol with subtilisin was determined and a ]( 1 value for
`benzyl alcoh ol with a-chymotrypsin was taken from the li tera(cid:173)
`ture . As can be seen in T able 1, benzyl a lcohol bind:; to the:;e
`enzymes much more poorly than does benzeneboroni c acid
`at its optimal pH. In addition, benzyl alcohol shows no pH de(cid:173)
`pendence in binding near pH 7 (11).
`Finally, we found no time d ependence in the apparent /{ 1
`values. This was shown first in t he millisecond range, using
`proflavin (8) in a stopped-flow a pparatus as an indicator for
`ch y motrypsin- inhibitor binding. The binding was too fa st to
`see and th e equilibrium profl rwin absorbance appea red im(cid:173)
`mediately after mixing. When an enzyme- inhibitor mixture
`was repeat edly assayed over a period of minutes, t he ap parent
`K 1 also remained constant. This was confirmed when it was
`noted th at the time course of inhibited subtilisin reactions, at
`low substra te concentration, was strictly first-order, as was
`the time course of chymotry psin reactions where t he inhibitor
`concentra tion was high enough to make the expression
`v =
`
`l1
`[8]
`"' [8] + 1\,. (1 + [I l/Kr)
`
`beeorne effectively
`
`.-\gain, if binding or debinding of th e inhibitor was slow , devia(cid:173)
`tion;; from first-order kinetics \rould have been observed. In
`
`5
`
`e
`'l::i
`"' 4
`"' 0
`-'
`
`0
`m-Nitro
`
`0
`p-Bromo
`
`0
`m- Amino
`unsubstituted
`
`3
`
`9
`
`8
`
`pKz
`
`7
`
`F rG. 2. Ha mmett-type plot of the subsl;ituent eiTect of the
`inhibi tion of subtilisin by benzeneboroni c acids. pK~ is derived
`fm m t he observed pH dependence of the inhibitions.
`
`this respect (unlike its pH dependence), the inhibitor behaves
`a:; a normal, substrate-like, bind ing agent .
`
`Bonm-11 n~odcl systen~ s tudies
`Boron-11 spectra of model systems were obtained since litera(cid:173)
`ture da ta show that, for boron t rifluorides and boric acid,
`complexation by a variety of lig;:l!lds prod uces a change in the
`1113 chemical shift of + 11- 13 ppm (12) . Thi s change may
`be associated with
`the ehang;e from
`trigonal
`to
`tetra(cid:173)
`hedral symmetry on com plexati on ( I 3) .
`\Ve observed a similar change in t he 1113 nmr po si tion~ on
`formation of the diethanolam ine t;o mplex of benzeneboronit;
`acid , and on addition of a n equim olar a moun t of imidazole to a
`m-nitrobenzeneboronic acid solut ion (Table 2) . In t he seco nd
`case, th e position of the I IJ:l nmr peak va ried continuously \rith
`composition between the chemical shi ft of t he pure boronic
`acid a nd t hat of th e eq ui molar mixture. In the case of th e eli(cid:173)
`ethanolamine complex, on the other hand, t\l·o peaks \rere
`obta ined, each at a fixed position, when a mixture of complex
`and free acid ester is present. These results ~uggest that in
`the case of the imidazole complex exchange i ~ b st on the nm r
`time scale. This is con:;istent wit h t he results obtained a bove
`with subtilisin and ch y m otr~r psin.
`
`DI SCUSSION ANU CONCLU S IONS
`Any reasonable interpretation of t he da.ta mu:;t explain both
`the pH dependence and t he rapidity of t he inhibition. Th e
`pH dependence indicates t hat a neu tral inhibitor .an~ :t
`neutral im.idazole side chain are involved. Sin ce the mhJlJJ(cid:173)
`tors are competitive with t he substrates used, this imidazole
`is presuma bly at the active site. Such an imidazole- boronJ r
`·
`.
`.
`-1 lo~rous to the
`·
`1 'l'l ·
`acid complex IS shown m model r
`11S 1s an. . "'
`·
`.
`imid azole- boronic acid complexes studi ed above. However,
`the existence of the nearby active-site serine suggests mode l
`B as a possibility. Analogous complexes \Yith an mmcle (14)
`
`TAB L E 2. nmr Studies of model systems
`
`Co mpound
`
`Dimethyl bem eneboronate
`D iethanolamine complex of benzene(cid:173)
`boroni c acid
`Dimethyl m-ni trobenzeneborona.te
`Imidazole complex of dimethyl m-ni tro(cid:173)
`benzeneboronate
`
`Chemi c:tl shifL *
`
`-U.5G ± 0.03 ppm
`
`8 . l '"i ± o.on ppm
`- 7. 55 ± 0. 1 ppm
`
`11 . g ± 0. 1 ppm
`
`* Chemi cal ~hift values were taken relaJive to met.hyl bora te.
`
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`

`
`480
`
`Biochemistry: Philipp and Bender
`
`or mrboxyl (15) ;z,'roup substituting for the imidazole are well
`known. However, the rate of formation and dissociation of
`such vomplexes is quite slow when boric acid and salicyl-
`amide (.l0l‘lV{l.l»iV(3S are used. This may be contrasted with the
`very rapid rates of inhibition observed here. This, taken to-
`gether with the “B nmr results, may indicate that imidazole
`coniplexution is alone sufficient to explain the inhibition.
`
`/OH
`OH
`©%B\~ W @B“ «~ 2 W
`
`OH
`
`—
`
`—
`
`Proc. Nat. Acad. Sci. USA
`
`2. Antonov, V. K., T. V. Ivaniva, I. V. Berezin, and K.
`iVI'a.rtinek, FEBS Lem, 7, 23 (1970).
`3. Bender, M. L., G.
`Clement, C. R. Gunter, and F. J.
`Kézdy, J. Amer. Chem. Soc., 86, 3697 (1964).
`4. Webb, J. L. , Enzymes and Metabolic I nh»ib2't0rs (Academic
`Press, New York, 1963), vol. I, pp. 150~1;')l.
`.3. Schwert, G. W., and Y. Takenaka, Biochim. Biophys.
`Acta, 16, 570 (_l9:'):")).
`6. Zerner, B., and M. L. Bender, J. Amer. Chem. Soc., 86,
`3674 (1964:).
`7. Polgar, L., and M. L. Bender, B'iochcm'zTst2'g/, 6, 610 (1967).
`8. Gla'/.er, A.-N., Proc. Nat. Acad. Sci. USA, 54, 171 (1965).
`9. Gla'/.er, A. N., J. Biol. Chcm., 242, 433 (1967).
`10.
`Juillard, J., and N. Gueguen, C. If. Acad. Sci. Paris, C,
`264, 259 (1967).
`11. Vztleir/Mela, P., and M. L. Bender, Biochemistry, 9, 2440
`(1970).
`in Progress in Boron Chemistry, ed. H.
`12. Schaeffer, R.,
`Steinberg, A. L. McClosl<ey (_MaeMillan, New York, 1964), vol.
`I, pp. 453—459.
`13. Niedenzu, K., and J. W. Dawson, Boron—N'itrogen Com-
`pounds (Academie Press, New York, 1965), pp. 156-158.
`14. Tanner, D. W., and T. C. Bruice, J. Amer. Chem. Soc., 89,
`6954 (1967).
`15. H. Schiifer, Z. Anorg. Allg. Chem, 250, 82 (1942).
`16. Bender, M. L., and F. C. Wedler Jr., J. Amer. Chem. Soc.,
`91, 3894 (1969).
`
`/H
`O
`
`.4
`
`0
`
`1;
`
`This work was supported by it grant from the National Insti-
`tutes of lleztlth. This paper was presented at the 8th Internation:L1
`(40“l§|'€-‘IS of Biochemistry, Interlziken, Switmrlztnd, September 4,
`1970. We also tlmnk Mr. Dan Netzel for performing the mnr
`:Ln21ly.~:e.~;.
`
`1'_ A"l‘““’Vr V. K., T. V. Ivzmivu, I. V. Berezin, and K.
`M:u'tmek, I)oIrl. Alaul. N(u47c. SSSR (Eng. transf), 183, 284 (1968).
`
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2137 - 7/7