Biochem. J. (1990) 267, 593-599 (Printed in Great Britain)

Chemical modification by pyridoxal 5'-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the P2phosphate-binding subsite of bovine pancreatic ribonuclease ARicardo M. RICHARDSON,*$ Xavier PARES* and Claudi M. CUCHILLO*t§*Departament de Bioquimica i Biologia Molecular, Facultat de Ciencies, and tlnstitut de Biologia Fonamental 'Vicent VillarPalasi', Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase Acould belong to the P2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemicalmodification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identifythese residues positively as part of the P2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstitutedderivatives, at Lys-l, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observedwhen either p1 or pa was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that bothsites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one ofthe sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonylgroup, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the P2 site blocked supports this hypothesis. Labelling of Lys-7 withpyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclicCMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatasethe kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higherKm and lower Vm were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and,hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blockinga secondary phosphate-binding site, namely P2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in thepresence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the P2phosphate-binding subsite.

INTRODUCTION

Bovine pancreatic RNAase A (EC 3.1.27.5) is an endonucleasethat hydrolyses single-stranded RNA when the base of thenucleotide in the 3'-position of the phosphodiester linkage is apyrimidine. Much evidence indicates that several binding subsitesof the enzyme are involved in its interaction with RNA (Richards& Wyckoff, 1971; Blackburn & Moore, 1982; de Llorenset al., 1989). Results of affinity-labelling experiments witha halogenated nucleotide analogue, 6-chloropurine riboside5'-monophosphate (cl6RMP), suggested the existence of aphosphate-binding subsite, called P2 (Pares et al., 1980a). Thehalogenated nucleotide specifically reacts with the enzyme to giveone major derivative, called derivative II, modified in the a-amino group of Lys-1, indicating that this region is close to, orbelongs to, a base-binding site (B3) and that the phosphate groupof the label binds to a positive cluster (P2). In order to explainthese results, a model for the interaction of RNA with RNAaseA was postulated (Fig. 1). Subsequent 1H-n.m.r. studies ofthe interaction between RNAase A and mononucleotides(Aru's et al., 1981, 1982) and dideoxynucleotides (Alonso etal., 1988) could be completely explained in terms of this model.Binding, kinetic and n.m.r. studies carried out by Irie et al.

(1984a,b, 1986) and the X-ray work ofMcPherson et al. (1986a,b)also lend support to the model and indicate its essentialcorrectness.

In more recent work (Richardson et al., 1988) it wasdemonstrated that the phosphate group of the nucleotide label ofderivative II remains bound to the P2 site. In the same work,studies on the interaction between RNAase A and naturalnucleotides confirmed that 3'-AMP binds to B2R2p2 whereas 5'-AMP binds to B2R2p1. In the present paper we describe com-parative chemical modification studies that demonstrate thephysical location of P2, by using derivative II and RNAase Aplus 3'-AMP as systems in which the P2 site is blocked. The maingoal was to achieve the labelling of Lys-7 and Arg-10, becauseprevious n.m.r. and molecular-model studies indicated that theseresidues were good candidates to be involved in P2 (Ards et al.,1981; de Llorens et al., 1989); moreover, they are conserved in allpancreatic RNAases (Beintema et al., 1988). We chose pyridoxal5'-phosphate (pyridoxal-P) and cyclohexane- 1,2-dione as chemi-cal reagents, since the former reacts with Lys-7 (Riquelme et al.,1975) and the latter with Arg-10 (Patty & Smith, 1975b).Differences in the chemical modification ofRNAase A, derivativeII, derivative E [identical with derivative II except that the labelis a nucleoside instead of a nucleotide (Alonso et al., (1986)] and

Abbreviations used and definitions: cl'RMP 6-chloropurine 9-fl-D-ribofuranosyl 5'-monophosphate; pyridoxal-P, pyridoxal phosphate; P-Pxy,5'-phosphopyridoxyl; derivative II, the main product of the reaction between RNAase A and cl6RMP, which has the nucleotide label attached tothe a-amino group of Lys-l; derivative E, the product of the reaction between RNAase A and 6-chloropurine riboside, which has the nucleosidelabel attached to the a-amino group of Lys-1; derivative A, [Na-(P-Pxy)-Lys-l]RNAase A; derivative B, [N-(P-Pxy)-Lys-7]RNAase A; derivative C,[N'-(P-Pxy)-Lys-4l]RNAase A.

t Present address: Department of Pharmacology, Northwestern University Medical School, 303 Chicago Avenue, Chicago, IL 60611, U.S.A.§ To whom correspondence should be addressed, at the Departament de Bioquimica i Biologia Molecular.

Vol. 267

593

R. M. Richardson, X. Pards and C. M. Cuchillo

Thr-45Phe- 1 20

B, Ser- 1 23

0

LP-6

Lys-66 R,

Fig. 1. Schematic diagram of the enzyme-substrate complex for RNAase A

B, R and P indicate base-binding, ribose-binding and phosphate-binding subsites respectively. There are specific recognition sites in the enzymefor each of the substrate moieties shown. B1 is specific for pyrimidine and B2 prefers purines. 3'-Pyrimidine mononucleotides bind to BIR,pl, 5'-purine mononucleotides bind to B2R2p1. 3'-AMP binds to B2R2p2. Derivative II and derivative E would have the labels occupying B3R3p2 andB3R3 respectively. The phosphate group of the phosphodiester bond hydrolysed by the enzyme binds to p1. The residues probably involved in eachsite are indicated.

RNAase A in the presence of 3'-AMP or 5'-AMP support theinvolvement of both Lys-7 and Arg-10 in the phosphate-bindingsubsite P2. Furthermore, we propose a model for the binding ofpyridoxal-P to RNAase A that explains the characteristics of itssubsequent reaction with Lys-7 and Lys-41.

MATERIALS AND METHODS

MaterialsBovine pancreatic RNAase (twice crystallized) was from

Biozyme (Blaenavon, Gwent, U.K.). L-1-Tosylamido-2-phenylethyl chloromethylketone-('TPCK'-)treated trypsin wasfrom Worthington Biochemical Corp. (Freehold, NJ, U.S.A.).2'(3')-CMP (mixture of isomers), 6-chloropurine riboside, 6-chloropurine riboside 5'-monophosphate, pyridoxal-P, NaBH4,3'-AMP, 5'-AMP, RNA, subtilisin, alkaline phosphatase, Trizmabase and dialysis sacs 250-7U were obtained from Sigma Chemi-cal Co. (St. Louis, MO, U.S.A.). Trifluoroacetic acid was fromFluka (Buchs, Switzerland). Constant-boiling-point 6 M-HCI waspurchased from Pierce Chemical Co. (Rockland, IL, U.S.A.).H.p.l.c.-grade acetonitrile and HCI were products fromFarmitalia Carlo Erba (Milan, Italy). (NH4)2CO3, NaCl, sodiumacetate, formic acid, H202, 2-mercaptoethanol and phenol wereobtained from Merck (Darmstadt, Germany). Cyclohexane- 1,2-dione was from Aldrich (Steinheim, Germany). CM-SepharoseCL-6B resin was obtained from Pharmacia Fine Chemicals(Uppsala, Sweden). 2',3'-Cyclic CMP (sodium salt) wassynthesized by the method of Szer & Shugar (1963).

Twice-distilled water filtered through a Norganic cartridgeand a 0.22 ,#m-pore-size membrane filter (Millipore Corp.,Bedford, MA, U.S.A.) was used in the h.p.l.c. experiments. The,uBondapak C,8 reverse-phase h.p.l.c. column (300 mm x 4 mm)and,Bondapak C Corasil stationary-phase pre-column were

purchased from Waters (Milford, MA, U.S.A.).

ApparatusAll h.p.l.c. experiments were carried out with a Waters modular

h.p.l.c. apparatus consisting of two model 6000 A pumps con-trolled by the model 680 automated gradient controller. Ad-ditional characteristics of the h.p.l.c. procedure are described inAlonso et al. (1986).

Methods

Purification of RNAase. Bovine pancreatic RNAase waspurified by the method of Taborsky (1959) to obtain the RNAaseA fraction.

Preparation of derivatives H and E. Derivative II was obtainedby the method described by Pares et al. (1980a). Derivative E wasobtained as described by Alonso et al. (1986).

Reaction of RNAase A and derivatives II and E with pyridoxal-P. The reaction between RNAase A and pyridoxal-P was carriedout by the method described by Riquelme et al. (1975) with someminor modifications. A 100 mg portion of RNAase A (91 gsM)and 5.5 mg of pyridoxal-P (0.27 mM) were dissolved in 80 ml of0.1 M-NaCl/10 mM-Tris/HCl buffer, pH 8.0. The reaction mix-ture was incubated at 4 °C in the dark with continuous stirring.After 3 min, 3 ml of a solution ofNaBH4 (20 mg/ml) was added.The mixture was then dialysed against 15 mM-Tris/HCl buffer,pH 8.0, to remove the pyridoxal-P that had not reacted, andchromatographed on a CM-Sepharose CL-6B column(1.5 cm x 45 cm) equilibrated with the same buffer. Elution wascarried out with a linear salt gradient (0-0.15 M-NaCl) in 15 mm-Tris/HCl buffer, pH 8.0. The fraction that contained the de-rivatives was dialysed against 15 mM-Tris/HCl buffer, pH 7.2,and rechromatographed on a CM-Sepharose CL-6B column(0.9 cm x 50 cm) equilibrated with the same buffer. Elution wascarried out with a 0-0.15 M-NaCl linear gradient in 15 mm-

1990

594

Chemical modification of RNAase A P2 phosphate-binding subsite

Tris/HCI buffer, pH 7.2. Reaction of pyridoxal-P with derivativeII and with derivative E was performed under the same con-ditions. For the reaction of RNAase A in the presence of naturalnucleotides the same procedure was followed except that either1.25 mM-5'-AMP or 1.25 mM-3'-AMP was added to the reactionmixture.The amounts of the unmodified RNAase A and of the different

derivatives obtained were calculated spectrophotometrically byusing values of 6278 9800 m-W cm-' for RNAase A (Sela &Anfinsen, 1957), 10 200 M-1W cm-' for derivative B, 9900 M-1W cm-'for derivative C (Borisova et al., 1974) and 11640 M-l cm-' forderivative A. Protein concentration was determined by means ofthe Bio-Rad assay kit (Bradford, 1976).

Reaction ofRNAase A with cyclohexane-1,2-dione. The reactionwas carried out according to the method described by Patty &Smith (1975a,b). A 25 mg portion of RNAase A was treated with2 ml of 50 mM-cyclohexane- 1,2-dione in 0.2 M-sodium boratebuffer, pH 9.0, at 37 °C, with continuous stirring. After 2 hincubation, 0.1 ml of cold 5 % (v/v) acetic acid was added to stopthe reaction. The mixture was subsequently dialysed against 1%(v/v) acetic acid in the cold for 48 h to remove the cyclohexane-1,2-dione that had not reacted and the excess of salt, and thenfreeze-dried. The same procedure was followed when either120 mM-3'-AMP or 120 mM-5'-AMP was added to the reactionmixture.

Assay of RNAase A activity. The RNAase A activity ofchromatographic fractions was determined spectrophoto-metrically by the method of Crook et al. (1960), with 2',3'-cyclicCMP as substrate.

Kinetic studies. Either 2',3'-cycic CMP or RNA was used as asubstrate. With 2',3'-cyclic CMP the substrate concentrationrange was 0.1-1.4 mm. The activity was measured by recordingthe increase in absorbance at 296 nm. The kinetic parametersfor RNA were determined with a concentration range of0.2-4 mg/ml. The decrease in absorbance at 300 nm wasmeasured. All assays were carried out in 0.1 M-sodiumacetate/HCl buffer, pH 5.5, containing 0.1 M-NaCl at 25 'C.The kinetic parameters were obtained by the non-linear-

regression data-analysis program ENZFITTER (Leatherbarrow,1987).Hydrolysis with subtilisin. The hydrolysis of RNAase A and

derivatives with subtilisin was carried out according to themethod of Richards & Vithayathil (1959).

Peptide analysis. Tryptic digestion and h.p.l.c. separationswere performed as previously reported (Alonso et al., 1986). Thedifferent enzymic samples were oxidized with performic acid bythe procedure of Hirs (1956). Amino acid analyses were carriedout as described by Vendrell & Aviles (1986).

RESULTS

Reaction of RNAase A with pyridoxal-P in the absence and inthe presence of natural nucleotides

We investigated the effects of the nucleotides 3'-AMP and 5'-AMP, which interact with B2R2p2 and B2R2p, respectively, onthe reaction between RNAase A and pyridoxal-P. Threederivatives of this reaction were obtained in a significant yield byfollowing the methodology indicated in the Materials andmethods section: derivative A corresponded to [N"-(P-Pxy)-Lys-l]RNAase A, derivative B corresponded to [N6-(P-Pxy)-Lys-7]RNAase A and derivative C was [Ne-(P-Pxy)-Lys-4l]RNAaseA. Their identity was confirmed by tryptic digestion and h.p.l.c.peptide analysis and is in agreement with previous reports (Raetz& Auld, 1972; Riquelme et al., 1975). Subsequently, the reactionwas carried out in the presence of 3'-AMP or 5'-AMP to blockspecifically P2 or p, respectively. The amounts of the differentderivatives obtained in each reaction are indicated in Table 1.The presence of a natural nucleotide did not significantly changethe yield of derivative A. However, the yields of both derivativesB and C were decreased by the presence of either nucleotide, 3'-AMP being more effective than 5'-AMP in preventing theformation of either derivative. These results support the involve-ment of both phosphate-binding sites p, and p2 in the reactionof pyridoxal-P with Lys-7 and Lys-41 but not in the reactionwith Lys-1.The kinetic parameters of RNAase A and the P-pyridoxyl

derivatives are indicated in Table 2. Derivative C is inactive witheither substrate, as could be expected from its modification inLys-41, an essential residue of the active site (Richards &Wyckoff, 1971). The kinetic parameters of derivative A are verysimilar to those of RNAase A for both substrates, indicating thatthe P-pyridoxyl moiety at Lys- I does not block any site importantfor either binding or catalysis. In contrast, derivative B showsaltered constants for both substrates. The decrease of both kcatand relative Vm8/([Eo] indicates that the label interferes in thecatalytic mechanism of both substrates. The increase in Km isdetected only in the case of RNA, suggesting that the binding ofthis substrate, but not that of 2',3'-cyclic CMP, is hindered by thepresence of this label.The covalently bound P-pyridoxyl group is a bulky label that

could interfere in different aspects of the substrate-binding andcatalytic mechanism. In order to clarify unambiguously the effectof the phosphate moiety of the label on the enzymic constants,derivative B was treated with alkaline phosphatase and thekinetic properties were determined again. No changes werefound in the constants of the treated derivative, suggesting thatthe phosphate group had not been removed by the phosphatase,a fact probably due to the tight binding of the phosphate to theprotein. The hydrolysis of the phosphate group was facilitated by

Table 1. Amounts of derivatives A, B and C and of unmodified RNAase A after reaction of RNAase A with pyridoxal-P in the presence and absence ofpurine nucleotides

A 100 mg portion of RNAase A (91 aM) was allowed to react with 5.5 mg of pyridoxal-P (0.27 mM) in a final volume of 80 ml at pH 8.0 and 4 °Cand the derivatives were separated as described in the Materials and methods section. The recoveries were calculated by using the 6278 values givenin the literature (see the Materials and methods section).

Derivative A Derivative B Derivative C RNAase ANucleotidepresent (mg) (%) (mg) (%) (mg) (%) (mg) (%)

None5'-AMP3'-AMP

20.5 10023.8 11619.1 93

18.59.57.1

10051.338.4

21 100 16.4 1007.9 35.7 38.6 2355.0 23.3 43.7 266

Vol. 267

595

R. M. Richardson, X. Pares and C. M. Cuchillo

Table 2. Kinetic parameters of RNAase A and derivatives A and B with 2',3'-cyclic CMP and RNA

In the experiments with RNA, relative values of VJ, /[EJ equal to 100{(JV../[EoJ)derivative}/{(Vna,c/[EoJ)RNAase A} were used. The values forRNAase A are taken as 100.

Km (mM for2',3'-cyclic CMP,

Enzyme Substrate mg/ml for RNA) kcat (min-') Relative V,n.I[Ej

2',3'-Cyclic CMP2',5'-Cyclic CMP2',3'-Cyclic CMP

RNARNARNA

82.69+7.864.41+1.2022.57+ 1.45

100102.5+ 1434.7+2.1

subtilisin cleavage of derivative B to the corresponding S-derivative B. Subsequently, the S-peptide B and S-protein B were

separated by h.p.l.c. The S-peptide B was treated with alkalinephosphatase and the resulting S-peptide B' was mixed withsaturating concentrations of S-protein to regenerate the S-derivative B'. Table 3 shows the kinetic parameters of RNAaseS, used as control, S-derivative B and S-derivative B'. Theimprovement of the catalytic constants for the S-derivative B' as

compared with S-derivative B indicates that the phosphate of theP-pyridoxyl moiety of the S-peptide B was effectively removed.The kinetic constants for RNAase S and S-derivative B are

similar to those previously discussed for RNAase A and de-rivative B (Table 2). The removal of the phosphate group of S-derivative B results in a new form, S-derivative B', that hadrecovered the same k,t. value as RNAase S with 2',3'-cyclicCMP as substrate. This suggests that the phosphate group of the[Ne-(P-Pxy)-Lys-7)RNAase A (derivative B) was occupying a siteclose to the catalytic centre, namely p1. The Km and Vm../[Eo]values for RNA also improve when the phosphate group isremoved. However, S-derivative B' still shows a poorer kineticperformance than RNAase S. This suggests that even in theabsence of the phosphate in the p1 site the binding and catalysisofRNA are still hindered by the presence of the pyridoxyl group

attached to Lys-7.

Reaction of pyridoxal-P with derivative II, derivative E andoxidized RNAase A

It is well established that cl6RMP reacts with RNAase A toyield derivative II, with the nucleotide label bound to the a-Natom of Lys-l (Pares et al., 1980a,b). The phosphate moiety ofthe bound nucleotide is located in the P2 phosphate-bindingsubsite (Richardson et al., 1988). On the other hand, reaction

of the corresponding nucleoside (6-chloropurine riboside) withRNAase A gives, as one of the major products, derivative E,identical with derivative II except that the phosphate group ismissing. We were interested in studying the effect of these labelson the reaction with pyridoxal-P. Thus the reactions ofpyridoxal-P with derivative II and with derivative E were carried out, withRNAase A as control. After each reaction a portion of themixture was directly oxidized with performic acid and digestedwith trypsin, without previous separation ofthe products. Trypticpeptides were analysed by reverse-phase h.p.l.c. The labelledpeptides were detected by monitoring the absorbance at 325 nm,

characteristic of pyridoxal-P, and at 254 nm, characteristic of thenucleotide and nucleoside. In the reaction with RNAase A threelabelled peptides were obtained (Fig. 2), which corresponded topeptides Lys-l-Lys-7 (I), Lys-l-Lys-10 (II) and Cys-40-Lys-61(III). This confirms that pyridoxal-P reacts at Lys-l, Lys-7 andLys41, as previously discussed. As shown in Fig. 2, each labelledpeptide appeared in split peaks. Amino acid analyses indicatedthat each peak of the doublet corresponded to the same peptide,suggesting that the different mobilities may be due to alterationof the label during the analytical process.The reaction of pyridoxal-P with derivative E modifies Lys-7

and Lys-41 with a yield of 60% and 70% relative to the controlreaction with RNAase A (Fig. 3). This indicates that the presence

of the nucleoside covalently bound to Lys-l partially interfereswith the reaction of pyridoxal-P with both Lys-7 and Lys-41. Onthe other hand, no P-pyridoxyl-peptide was detected in thereaction between pyridoxal-P and derivative II. For both de-rivative II and derivative E the lack of reaction on Lys-l was

already expected, as this residue is covalently modified by a

nucleotide and a nucleoside respectively. Differences in thereaction at the other two lysine residues should be exclusively

Table 3. Kinetic constants of RNAase S, S-derivative B and S-derivative B'

Relative V.../[E(J is defined in Table 2.

Km (mM for2',3'-cyclic CMP,

Enzyme Substrate mg/ml for RNA) kcat (min 1) Relative Vma../[EI

RNAase SS-derivative BS-derivative B'

RNAase SS-derivative BS-derivative B'

2',3'-Cycic CMP2',3'-Cyclic CMP2',3'-Cyclic CMP

RNARNARNA

0.49 ±0.030.50+0.130.54+0.11

0.54+0.041.40+0.080.98+0.02

51.86+1.132.16+0.948.66+1.4

10042+ 1.367+7.7

1990

RNAase ADerivative ADerivative B

RNAase ADerivative ADerivative B

0.42+0.030.46+0.020.50+0.05

0.65+0.060.62+0.040.98+0.10

596

Chemical modification of RNAase A p2 phosphate-binding subsite

0.50 t (a)

0.25 1

0.4

I-,

"W0.2 °_

0.50 - (b)

0.251 -

0O

LIi

25 50Time (min)

1,I 1IJ

a a--

JLL~LLJlJ0 25 50

Time (min)

Fig. 2. Elution profile of the separation by reverse-phase h.p.l.c. of thetryptic peptides of RNAase A (a) and the product of reactionbetween RNAase A and pyridoxal-P (b)

Amino acid analysis showed that peptide I is Lys-l-Lys-7, peptideII is Lys-l-Lys-10 and peptide III is Cys-40-Lys-61.

attributed to the presence of the phosphate group in thenucleotide label of derivative II, which is absent from derivativeE. The presence of this phosphate group, which occupies p2, as

demonstrated by Richardson et al. (1988), completely suppresses

the reaction of pyridoxal-P with both Lys-7 and Lys-41.Finally, the formation of derivatives is completely -sup-

pressed when pyridoxal-P is incubated with performic acid-oxidized RNAase A. This emphasizes the key role of thephosphate-binding subsites in the reaction of pyridoxal-P withRNAase A.

Effects of 3'-AMP and 5'-AMP on the modification of Arg-10by cyclohexane-1,2-dione

In addition, or as an alternative, to Lys-7, Aru's et al. (1981)and de Llorens et al. (1989) proposed Arg-10 as a possiblecomponent of the p2 phosphate-binding subsite (Fig. 1) on thebasis of molecular models. Studies by chemical modification ofArg-10 have been hampered by its low reactivity (Takahashi,1968; Ijima et al., 1977). Only cyclohexane-1,2-dione reacts in a

significant amount with this residue; Arg-39 and Arg-85 are alsopartially modified (Patty & Smith, 1975b). In the present workwe used the methodology of those authors to label Arg-10. Fig.4 shows the reverse-phase h.p.l.c. separation of the trypticpeptides obtained after reaction of RNAase A and cyclohexane-1,2-dione. The reaction at Arg-10 was quantified by measuringthe amount of peptide Phe-8-Arg-10. About 37% of RNAase Awas found to be modified at this arginine residue under theconditions used. When the reaction was carried out in the

presence of 5'-AMP, about 30% of the protein was labelled atArg-10 (19% inhibition with respect to the reaction without

Vol. 267

Fig. 3. Elution profile of the separation by reverse-phase h.p.l.c. of thetryptic peptides of derivative E (a) and the product of reactionbetween derivative E and pyridoxal-P (b)

Peptide 1 corresponds to Lys-l-Lys-7 with the nucleoside label atthe a-amino group of Lys-l; peptide 2 corresponds to Lys-l-Arg-10, with the nucleoside label at the a-amino group of Lys-l and theP-pyridoxyl group at Lys-7; peptide 3 is peptide Cys-40-Lys-61,with the P-pyridoxyl label at the e-amino group of Lys-41.

nucleotide), and a complete lack of modification was observedwhen 3'-AMP was added (Fig. 4). Therefore 3'-AMP, whichinteracts in p2., fully protects Arg-10 against modification bycyclohexane- 1 ,2-dione, suggesting that this residue must be closeto P2-

DISCUSSION

The reaction of c16RMP with the a-amino group of Lys-1 toyield derivative II served to postulate the existence of the P2phosphate-binding subsite of RNAase A (Pares et al., 1980a,b).Further work of our group (Aru's et al., 1981, 1982; Alonso et al.,1988; Richardson et al., 1988) provided more evidence on theexistence of P2. In the present work we have investigated thephysical localization of the P2 subsite, i.e. the amino acid residuesinvolved in phosphate-binding. Good candidates for this siteshould be positively charged residues some 0.9-1.1 nm distantfrom Lys-I (de Llorens et al., 1989). Molecular models of theRNAase A-cl6RMP interaction indicated- that residues Lys-7and Arg-10 were at the right distance to bind the phosphatemoiety of the nucleotide (Arus et al., 1981; Richardson et al.,1988). The study of a complex of the enzyme with thepentanucleotide pApUpApApG by means ofmodel building andcomputer graphics also suggested that Lys-7 and Arg-10 werelocated in P2 (de Llorens et al., 1989). Moreover, both residuesare 100% conserved in all known pancreatic RNAases [40

Time (min)

0

* 0.25

- 1- -

75

0.4

";(

0.2 _

0

0.4

0.2 0

0.2o

50Time (min) 75

I

111HItI

I I11

597

R. M. Richardson, X. Pares and C. M. Cuchillo

(a) (pi)Lys-41

Time (min)

0.4

(b) (p1)Lys-41

0.2 °"

0

0.4

0.2 So

LLys-7 (P2)

Fig. 5. Model for the interaction and covalent reaction of pyridoxal-P andRNAase A

(a) Formation of derivative B by interaction of the phosphate groupwith Lys-7 and reaction of the aldehyde group at Lys-41. (b)Formation of derivative C by interaction of the phosphate group atLys-41 and reaction of the aldehyde group with Lys-7.

0

50Time (min)

Fig. 4. Elution profile of the separation by reverse-phase h.p.l.c. of thetryptic digest of the product of reaction between RNAase A andcyclohexane-1,2-dione

Peptide 1 corresponds to Phe-8-Arg-10. (a) Incubated in the absenceof nucleotide; (b) incubated with 5'-AMP in the reaction mixture;(c) incubated with 3'-AMP in the reaction mixture. See the Materialsand methods section for more details.

species (Beintema et al., 1988)], suggesting that they have an

important role in the structure and function of the enzyme.To demonstrate further the involvement of these residues in P2,

specific chemical modifications in the presence and in the absenceof groups that bind to P2 were carried out. The reaction withpyridoxal-P (Riquelme et al., 1975) was used for the modificationat Lys-7. The reaction was carried out in the presence of either5'-AMP (binding at B2R2p1) or 3'-AMP (binding at B2R2p2) inorder to block specifically p1 and P2 respectively. The resultsshown in Table 1 indicate that the correct binding of pyridoxal-P to RNAase A requires that both centres p1 and P2 are free inorder to form a complex, which then reacts in a specific manneryielding the covalent derivatives B and C. The kinetic constantsfound for derivative B also suggest that the P-pyridoxyl groupoccupies several subsites. When the low-molecular-mass substrate2',3'-cyclic CMP is used Km is unchanged whereas kc,a is

significantly decreased (Table 2). However, when the phosphategroup of the corresponding S-peptide B is removed by alkalinephosphatase, yielding S-peptide B', the kcat value of thereconstituted S-derivative B' is almost as high as that of nativeRNAase A. This fact can be explained by postulating that thephosphate moiety of derivative B is occupying p1, thus affectingthe kinetics of 2',3'-cyclic CMP hydrolysis. Removal of thephosphate group reverts the kinetics with that substrate back tonormal. S-derivative B', however, does not recover the RNAaseA kinetic values with RNA, indicating that the binding of thissubstrate needs an additional subsite that is still blocked evenafter removal of the phosphate of S-derivative B. From theseexperiments we postulate that the pyridoxal moiety of [N6-(P-Pxy)-Lys-7]RNAase A is located in P2 while its phosphate moietyoccupies p1.

We have also demonstrated that the presence of a phosphatemoiety in P2, such as in derivative II, suppresses the covalentmodification by pyridoxal-P at Lys-7. Similarly, the reaction onLys-41 is also prevented when P2 is blocked, suggesting that thephosphate group of pyridoxal-P should interact in P2 before theSchiff base could be formed at Lys-41. This indicates that a freeP2 phosphate-binding subsite is absolutely necessary for thereaction of pyridoxal-P with either lysine residue.

These results can be explained by postulating that the reactionof pyridoxal-P with RNAase A follows the model proposed inFig. 5. Thus the phosphate group of the label must interact in P2(near Lys-7) when the covalent modification takes place in Lys-

1990

0.50

- 0.25

0

(P2)

Time (min)0.50

, 0.25

598

Chemical modification of RNAase A P2 phosphate-binding subsite

41, and the phosphate group of pyridoxal-P must interact in Lys-41 (p1) when the reaction is at Lys-7.The absence of disubstituted derivatives as products of the

reaction and the observations by Raetz & Auld (1972) that onlya single molecule of pyridoxal-P interacts per molecule ofRNAase A and that pyridoxal-P is unable to inactivate RNAasecompletely support the model.

This model would explain why pyridoxal (without phosphate)does not react at all with RNAase A (Means & Feaney, 1971;Raetz & Auld, 1972) and also why no reaction is detectedwith pyridoxal-P when the phosphate-binding subsites aredisorganized such as in the case of oxidized RNAase A.From the above discussion it is reasonable to assume that Lys-

7 is involved in the P2 phosphate-binding subsite. Several previousreports support this role for Lys-7. Thus experiments witholigomeric substrates and an RNAase S' with a modified S-peptide ([7-norleucine]S-peptide) clearly support the involvementof Lys-7 in the interaction with the phosphate group that bindsin P2 (Irie et al., 1986). Detailed X-ray-crystallographic studies ofa complex between RNAase A and d(pA)4 indicate that thephosphate adjacent to that occupying p1, i.e. p2, is at 0.47 nmfrom Lys-7, to which it is apparently salt-bridged (McPhersonet al., 1986 a,b).The role of Arg-10 is more obscure because of the difficulty of

specifically labelling this residue. However, all reports indicate asignificant decrease of activity towards RNA when this arginineresidue is modified. Thus the replacement of arginine by ornithinein the [Orn10]S-peptide results in a 55 % decrease in the activityof the corresponding RNAase S' towards RNA (Marchiori etal., 1972). Modification of Arg-10 by cyclohexane-1,2-dionesignificantly decreases the activity of RNAase A towards RNA.Such a loss of activity was not recovered after treatment withhydroxylamine (Patty & Smith, 1975b). Reaction of Arg-10 withketoxal also results in a decreased activity towards RNA (Ijimaet al., 1977). In the present study it was demonstrated that thereaction of cyclohexane-1,2-dione with Arg-10 is completelyblocked by the presence of 3'-AMP, whose phosphate groupspecifically binds at P2 (Fig. 4). In contrast, reaction at thisresidue is only slightly diminished by the presence of 5'-AMP(which has its phosphate group bound at p1). This is also clearevidence that Arg-10 is very probably involved in the phosphate-binding at P2. Modification of Arg-10 would yield an enzymewith an altered P2 subsite, which would explain its low activitytowards RNA (Ijima et al., 1977).

In summary, the combined chemical-modification studies withpyridoxal-P and cyclohexane- 1,2-dione, both in the absence andin the presence of 5'-AMP and 3'-AMP, strongly support theinvolvement of Lys-7 and Arg-10 in the p2 phosphate-bindingsubsite of RNAase A.

R. M. R. was a recipient of a fellowship from the Instituto deCooperaci6n Iberoamericana. This work was supported by Grant PB85-

0097 from the Comisi6n Interministerial de Ciencia y Tecnologi'a of theMinisterio de Educaci6n y Ciencia (Spain). We thank the Fundaci6M. F. de Roviralta (Barcelona) for grants for the purchase of equipment.

REFERENCESAlonso, J., Nogues, M. V. & Cuchillo, C. M. (1986) Arch. Biochem.

Biophys. 246, 681-689Alonso, J., Paolillo, L., D'Auria, G., Nogues, M. V. & Cuchillo, C. M.

(1988) Int. J. Pept. Protein Res. 31, 537-543Aris, C., Paolillo, L., Llorens, R., Napolitano, R., Pares, X. & Cuchillo,

C. M. (1981) Biochim. Biophys. Acta 660, 117-127Arus, C., Paolillo, L., Llorens, R., Napolitano, R. & Cuchillo, C. M.

(1982) Biochemistry 21, 4290-4297Beintema, J. J., Schuller, C., Irie, M. & Carsana, A. (1988) Prog.

Biophys. Mol. Biol. 51, 165-192Blackburn, P. & Moore, S. (1982) Enzymes 3rd Ed. 15, 317-433Borisova, S. N., Matrosov, V. J., Shliapnikov, S. V. & Karpeisky,M. Ya. (1974) Mol. Biol. (Moscow) 8, 286-295

Bradford, M. M. (1976) Anal. Biochem. 72, 248-254Crook, E. M., Mathias, A. P. & Rabin, B. R. (1960) Biochem. J. 74,234-238

de Llorens, R., Aris, C., Pares, X. & Cuchillo, C. M. (1989) Protein Eng.2, 417-429

Hirs, C. H. W. (1956) J. Biol. Chem. 219, 611-621Ijima, H., Patrzyc, H. & Bello, J. (1977) Biochim. Biophys. Acta 491,264-272

Irie, M., Watanabe, H., Ohgi, K., Tobe, M., Matsumura, G., Arata, Y.,Hirose, T. & Inayama, S. (1984a) J. Biochem. (Tokyo) 95, 751-759

Irie, M., Mikami, F., Monma, K., Watanabe, H., Yamaguchi, R. &Nagase, H. (1984b) J. Biochem. (Tokyo) 96, 89-96

Irie, M., Ohgi, K., Yoshinaga, M., Yanagida, T., Okada, Y. & Teno, N.(1986) J. Biochem. (Tokyo) 100, 1057-1063

Leatherbarrow, R. J. (1987) ENZFI'TTER: A Non-Linear RegressionData Analysis Program for the IBM-PC, Elsevier Biosoft, Cambridge

Marchiori, F., Borin, G., Moroder, L., Rocchi, R. & Scoffone, E. (1972)Biochim. Biophys. Acta 257, 210-221

McPherson, A., Brayer, G. D. & Morrison, R. D. (1986a) Biophys. J. 49,209-219

McPherson, A., Brayer, G. D. & Morrison, R. D. (1986b) J. Mol. Biol.189, 305-327

Means, G. E. & Feaney, R. E. (1971) J. Biol. Chem. 246, 5532-5533Pares, X., Llorens, R., Arns, C. & Cuchillo, C. M. (1980a) Eur. J.

Biochem. 105, 571-579Pares, X., Puigdomenech, P. & Cuchillo, C. M. (1980b) Int. J. Pept.

Protein Res. 16, 241-244Patty, L. & Smith, E. L. (1975a) J. Biol. Chem. 250, 558-564Patty, L. & Smith, E. L. (1975b) J. Biol. Chem. 250, 565-569Raetz, C. R. H. & Auld, D. S. (1972) Biochemistry 11, 2229-2236Richards, F. M. & Vithayathil, P. J. (1959) J. Biol. Chem. 234, 1459-1465Richards, F. M. & Wyckoff, H. W. (1971) Enzymes 3rd Ed. 4, 647-806Richardson, M. R., Pares, X., Llorens, M., Nogues, M. V. & Cuchillo,

C. M. (1988) Biochim. Biophys. Acta 953, 70-78Riquelme, P., Brown, W. E. & Marcus, F. (1975) Int. J. Pept. Protein

Res. 7, 379-387Sela, M. & Anfinsen, C. B. (1957) Biochim. Biophys. Acta 24, 229-235Szer, W. & Shugar, D. (1963) Biochem. Prep. 10, 139-144Taborsky, G. (1959) J. Biol. Chem. 234, 2652-2656Takahashi, K. (1968) J. Biol. Chem. 243, 6171-6179Vendrell, J. & Aviles, F. X. (1986) J. Chromatogr. 358, 401-413

Vol. 267

Received 20 June 1989/6 November 1989; accepted 16 November 1989

599