Hiratake, Jun

Kato, Hiroaki

Oda, Jun'ichi

Kyoto University Research Information Repository

TitleMechanism-Based Inactivation of Glutathione Synthetase byPhosphinic Acid Transition-State Analogue (MOLECULARBIOFUNCTION - Functional Molecular Conversion)Author(s)Hiratake, Jun; Kato, Hiroaki; Oda, Jun'ichiCitationICR annual report (1995), 1: 44-45Issue Date1995-03URL http://hdl.handle.net/2433/65658RightType ArticleTextversionpublisherKyoto University44 IeR Annual Report, Vol. I, 1994Mechanism-Based Inactivation of Glutathione Synthetaseby Phosphinic Acid Transition-State Analoguelun Hiratake, Hiroaki Kato, and lun'ichi OdaA potent and time-dependent inactivator of glutathione synthetase was synthesized. According to the proposedreaction mechanisms of glutathione synthetase, we designed and synthesized the phosphinate-type transition-state analogue 1. The (S, R)-isomer of I having the same relative configurations as y-Glu-L-Cys was found to bea potent and time-dependent inactivator of this enzyme with an inhibition constant Ki of21 nM, while the (S, S)-lrelated to y-Glu-D-Cys was practically inactive. The time-dependent inactivation occurred only when ATP waspresent, with the onset rate of inactivation (kon =8.29 sec- 1 mM- l ) being 75-times slower than the enzymaticreaction, and the inactivated enzyme slowly regained its activity (tll2=53hr). The molecular basis for thisinactivation was probed by an X-ray crystallography of the enzyme complexed with (S, R)-l and ATP. The X-ray diffraction analysis has shown that a mechanism-based inactivation was operative in which the phosphinateoxygen (P-O-) of 1 was phosphorylated by ATP within the enzyme active site to form ADP and aphosphorylated 1 which is highly analogous to the proposed transition state.Keywords: Glutathione synthetase/ Transition-state analogue/ Phosphinate/ Time-dependentinactivation/ Mechanism-based phosphorylation/ coo' CH,~H ).~I;;ATI' (J l o·y.GI".C)', • '/"'_ 11./ IIi N/" Co" C-O-r'-O') II II"o a 0(I)Scheme Iy-Glu,C)'l·GI)' t- ADI' .- 1',(l;llJIHhioflc)Glutathione synthetase (GSHase, EC 6.3.2.3), cata-lyzing the ligation of y-Glu-Cys and glycine with the aid ofATP, is a key enzyme in glutathione biosynthesis. Thedetailed reaction mechanisms, however, are yet to be wellunderstood, although the X-ray crystal structure of thisenzyme has recently been defined [1]. In the light ofmechanistically well-studied other ligases such asglutamine synthetase [2] or D-Ala-D-Ala ligase [3], thereaction catalyzed by GSHase is thought to proceedthrough the initial formation of a putative acyl phosphateintermediate (I), followed by nucleophilic attack ofglycine to yield glutathione, ADP and inorganicphosphate (Scheme 1).GlyCOO· CH,CH)•)"'~ II A /CH,CH,COO'H N CoN • P) (.I) II 11 ....... 0-o 01 a . (5. "IS)1b : (5. R)1 C : (S. 5)MOLECULAR BIOFUNCTION -Functional Molecular Conversion-InstructorTANAKA,Takuji(D Agr)InstructorHIRATAKE,Jun(D Agr)InstructorKATO,Hiroaki(D Agr)Assoc ProfNISHIOKA,Takaaki(D Agr)ProfessorODA,Jun'ichi(D Agr)Scope of researchOur research aims are to analyze structure-function relationships of biocatalysts in combination with organicchemistry, structural biology and computer science, and to apply biocatalysts to stereospecifc organic sysnthesis.Major subjects are the design and preparation of antibodies catalyzing stereoselective reactions, the reactionmechanisms of glutathione synthetase from E. coli with static and time-resolved X-ray crystallography, the mode ofaction of lipase-activating protein, crystallographic analysis of asparagine synthetase and y-L-glutamyl-L-cysteinsynthetase, and creation of enzymatic reaction database and development of algorithms for protein sequence analysis.Students:SUYAMA, Mikita (DC)HARA, Takane (DC)NAKATSU, Toru (DC)SHIBATA, Hiroyuki (DC)KATO, Makoto (MC)IMAEDA, Yasuhiro (MC)MATSUDA, Keiko (MC)45COO· CH,CH)) I' coo+ "'·../"C-~/--....P,/'-/ 0H)N II II'O-p/o 0 11'0'o1 bFigure 2. The Fa-Fe electron density surface of the pho-sphorylated phosphinate 1b and MgADP, superimposed withrefined structure of the active site region. The contributions ofthe phosphorylated 1b and MgADP were omitted from the Fecalculation. The contour level is 3.56.most likely to reflect a mechanism· based phosphorylationof the phosphorus oxyanion (PO-) of 1b by ATP withinthe enzyme active site.If this is the case, then the tightly boundphosphorylated inhibitor 1b and ADP should be visiblewith an X-ray diffraction analysis of GSHase complexedwith 1b and ATP. This in fact proved to be the case: theelectron density map around the active site has clearlyshown that the y-phosphate of ATP has been transferredto the inhibitor phosphorus oxyanion (PO-) to form aphosphorylated 1b and ADP within the enzyme active site(Figure 2). Since the phosphorylated 1b is highlyanalogous to the proposed transition state, thephosphinate 1 serves as an excellent probe for definingwhat the real transition state is like and how this enzymestabilizes it to facilitate the reaction.References1. Kato H, Tanaka T, Yamaguchi H, Hara T, NishiokaT, Katsube Y and Oda J, Biochemistry, 33,4995-4999(1994).2. Meister A, In The Enzymes, Boyer P.D., Ed.;Academic Press, New York, 1974; Vol. 10, pp.699-754.3. Mullins L.S., Zawadzke L.E., Walsh C.T. andRaushel F.M., 1. Bioi. Chern., 265,8993-8998 (1990).4. Hiratake J, Kato Hand Oda J, J. Am. Chern. Soc.,116, 12059-12060 (1994).5. Morrison J.F. and Walsh c.T., In Advances inEnzymology; Meister A., Ed.; John Wiley: NewYork, 1988; Vol. 61, pp.201-301.7060 [la) =0 >,M 0.4 11M50i 402:(l.1.0 ~IM0 30<:20 2.011 M10 4.0 11M2 6 10 12 14 16Time (min)Figure 1. Progress curves for the inactivation of GSHase by 1a.The reaction was initiated by adding enzyme to an assay mixturecontaining y-Glu-Cys (0.2 mM), Gly (15 mM), ATP (5 mM) and1a (0.2-4,uM) in 50 mM Tris-HCI (pH 7.5) at 37'CThe inactivated enzyme slowly regained its activity(tl/2=53 hr) when incubated for several days, but notwithin a steady-state time scale, thus the inhibition beingpractically irreversible. The onset rate of inactivation(kon) [5] was calculated from the progress curves and wasfound to be 8.29s- I mM- 1 with 1a. This value,compared with the kinetic constants for y-Glu-Cys[koIKm =625s- ImM- 1], means that the inactivationprocess is 75 times slower than the enzymatic reaction.Steady-state kinetic analysis revealed that the inhibitionwas competitive with y-Glu-Cys, with the inhibitionconstant K; being 53 nM with 1a. As expected, the (S,R)-lb having the same relative configuration as y-Glu-L-Cys was found to be an extremely potent and time-dependent inactivator (K;=21 nM), whereas the (S, S)-isomer 1c related to D-CyS showed only 18% inhibition at39mM.The molecular basis for the time- and ATP-dependentinactivation by 1 was examined. Considering that theenzyme inactivation was observed only when both ATPand phosphinate 1 were present, the inhibition pattern isWe therefore designed the transition-state analogue 1in which the C-terminal carboxyl group is replaced by atetrahedral phosphinyl .group with a 2-carboxyethylmoiety mimicking the incoming glycine, and used thephosphinate 1 to probe the reaction mechanisms of thisenzyme. Starting with racemic, (R)-, and (S)-l-(amino-propyl)phosphinic acid derivative [4], the diastereomericmixture (S, RIS)-la and each diastereomer (S, R)-lb and(S, S)-lc were synthesized.The phosphinate 1a was found to be a remarkablypotent inactivator of GSHase. Treatment of GSHasewith 1a resulted in time-dependent inactivation of the. enzyme as shown by the progress curves (Figure 1).Interestingly, no inactivation was observed without ATP,whereas complete inactivation resulted when both 1a andATP were present.

Mechanism-Based Inactivation of Glutathione Synthetase by Phosphinic Acid Transition-State Analogue (MOLECULAR BIOFUNCTION - Functional Molecular Conversion)

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Mechanism-Based Inactivation of Glutathione Synthetase by Phosphinic Acid Transition-State Analogue (MOLECULAR BIOFUNCTION - Functional Molecular Conversion)

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