Understanding enzyme action at solid surfaces

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					                                                                                                                                                             Biocatalysis   309

Understanding enzyme action at solid surfaces
P.J. Halling1
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, Scotland, U.K.

               In solid-phase synthesis, there is interest in using enzymes that normally act on dissolved substrates. It is
               normally observed that rates and yields are substantially reduced when the usual substrates are covalently
               attached to a solid particle. Recently, there has been some progress in understanding the reasons for this,
               and hence how to improve behaviour. Diffusion of enzyme molecules into some of the support particles
               used in solid-phase chemistry is slow or absent. Methods are now available to visualize the sites of reaction,
               and hence detect this problem, and identify better support materials. Chemical equilibrium positions for
               reactions at the surface can be substantially altered compared with those in solution, so may unexpectedly
               limit yields. The shift can also be exploited to carry out, for example, direct synthesis of peptide bonds in
               an aqueous environment. The rate of enzyme attack depends on how the substrate moiety is attached to
               the surface, with an optimal ‘spacer’ length.

Introduction                                                                                  systems, for example when used to assay activity. The present
Most enzyme-catalysed reactions involve substrates that are                                   paper will deal with three questions that have been addressed
dissolved in the reaction medium. Some enzymes, such as                                       in work at Strathclyde (in collaboration with others). Can
cellulases and amylases, naturally act at the surface of insol-                               the enzyme reach the sites of reaction in a porous support
uble substrates, and have special features to facilitate this. But                            particle? How will the equilibrium position of the reaction
there is increasing interest in enzymes that normally have                                    be affected? What is the influence of the length of a ‘spacer’
dissolved substrates acting instead on groups attached to solid                               attaching the substrate moiety to the surface?
supports. These systems can be seen as complementary to the
more familiar immobilized enzymes and indeed have been
referred to as immobilized substrates.                                                        Accessibility of enzyme to sites
   Enzyme action on surface-attached substrates is relevant in                                within beads
a number of situations. In solid-phase synthesis, the specifi-                                An obvious first condition for catalysis is that the enzyme
city of enzymes can be valuable [1,2], particularly for re-                                   molecules reach the sites at which substrate moieties are
moving or avoiding the need for protecting groups, or                                         attached. In solid-phase chemistry, there is a natural pref-
selectively cleaving linkers in a final step. Where combi-                                    erence for support materials with a high density of sites for
natorial libraries of potential enzyme substrates are prepared                                attachment of the species being synthesized. This is achieved
on solid supports, it is convenient to be able to assay them                                  by using support materials with very high surface areas, as a
while still attached [3–6]. Abnormal action on insoluble                                      consequence of very small pore sizes, or even with a mobile
substrates may also be relevant in certain disease states                                     gel-type network without well-defined pores. Thus there is
[7–9], such as attack on cartilage in arthritis and cell surfaces                             a clear possibility that enzyme molecules will not be able
in pancreatitis.                                                                              to penetrate all parts of the support bead, or at least their
   When enzymes are employed in solid-phase synthesis, it                                     diffusion through the particle will be greatly hindered. One
is normally observed that rates and yields are substantially                                  type of particle that has been particularly recommended for
reduced when the usual substrates are covalently attached to                                  enzyme reactions are the PEGA [poly(ethylene glycol) acryl-
a solid particle. Enzyme assays on solid-phase substrates are                                 amide] range [11]. These consist of a core acrylamide structure
usually used just to give relative rates, but where absolute ac-                              to which poly(ethylene glycol) chains are attached. The beads
tivity has been measured, it is again low compared with those                                 swell in both water and a range of organic solvents, and permit
in solution. There have recently been some initial approaches                                 high loadings of reactive sites at approx. 200 µmol · g−1 .
to improve understanding of how enzymes behave in such                                        There is evidence that PEGA beads may restrict penetration
a system [10]. Better understanding should be the basis for                                   of enzyme molecules, particularly larger ones. One of the
designing reaction systems to obtain better rates and yields.                                 clearest demonstrations used confocal Raman microscopy to
It will also aid the interpretation of results obtained in such                               monitor reaction in the bead interior [12].
                                                                                                 An alternative method to image the sites of enzyme reac-
Key words: confocal microscopy, fluorophore, poly(ethylene glycol) acrylamide (PEGA), solid-   tion is to use fluorescence microscopy. Ideally, we would like
phase synthesis, surface chemistry.
                                                                                              to use optical sectioning of intact beads, to avoid possible arti-
Abbreviations used: Ac, acetyl; CPG, controlled pore glass; PEGA, poly(ethylene glycol)
acrylamide.                                                                                   facts from sample preparation. Confocal fluorescence micro-
  email                                                              scopy is not normally applicable to these systems, however,

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310   Biochemical Society Transactions (2006) Volume 34, part 2

      Figure 1 Spatially resolved time course of thermolysin action on          Scheme 1 Equilibrium positions for coupling of saturated
      immobilized substrate in PEGA beads                                       aqueous Fmoc (fluoren-9-ylmethoxycarbony)-Gly to free and
      Fmoc (fluoren-9-ylmethoxycarbonyl)-Phe-Phe-PEGA beads were ex-             immobilized phenylalanine residues
      posed to enzyme for the times shown, then amino groups released were      Reaction at bulk pH 7.4 was catalysed by thermolysin, with PEGA1900 as
      labelled with dansyl. Optical sections of intact beads were examined by   solid phase. Data from [15].
      two-photon fluorescence microscopy. Reproduced from [14]. c 2003
      The Royal Society of Chemistry.

                                                                                reaction at different positions in the optical section through
                                                                                the bead. This has proved quite challenging to implement as a
                                                                                quantitative assay, but we are now close to success ( J. Deere,
                                                                                A. Lalaouni, B. Maltman, G. McConnell, J. Girkin, S.L.
                                                                                Flitsch and P.J. Halling, unpublished work).

                                                                                Equilibrium of reactions on solid-attached
      as the high fluorophore concentrations absorb excitation                  substrates
      light before it can reach deep into the bead. As a result, the            On first thought, it might be expected that attachment to
      fluorescence detected from the bead core is ‘artifactually’ low.          a solid support would have little effect on the equilibrium
      Instead, we have used two-photon fluorescence microscopy                  of a reaction, provided the medium stays the same (e.g.
      for this purpose. In this approach, light absorption is largely           aqueous). In fact, however, there can be a substantial shift in
      restricted to the focal point as this is scanned through the              equilibrium position, for example making peptide synthesis
      bead, because only there is the intensity high enough for                 largely favourable even in an aqueous environment (e.g.
      substantial two-photon absorption events. We were able to                 Scheme 1; [15,16]). This can be exploited preparatively,
      show that two-photon microscopy gave fairly good quantifi-                but may also be the reason for poor yields observed in
      cation of fluorophore concentrations throughout the bead,                 some attempted hydrolysis reactions on peptide and other
      after correction for an aberration effect as a function of depth          substrates.
      into the sample [13].                                                        Three factors may contribute to the shift in equilibrium
         A first approach to identify where the enzyme was acting               [16].
      used a post-reaction labelling method [14]. Enzyme-catalysed                 (i) With one component of a coupling reaction attached to
      hydrolysis of a peptide bond liberated a free amino group                 the solid phase, it is easier to add and remove a large excess
      on the substrate portion still bound to the bead. After                   of the other. This is of course theoretically trivial, although a
      stopping the enzyme reaction at various times, the free amino             practically useful feature of solid-state chemistry. However,
      groups were labelled by reaction with dansyl chloride. After              it is not the explanation for some observed shifts, such as
      washing, imaging of the positions of the dansyl fluoro-                   the example above, because this mass action effect has been
      phore revealed where the enzymatic reaction had occurred                  allowed for.
      (Figure 1). This indicated that the reaction in the bead core                (ii) If the soluble reaction component contains hydro-
      lagged significantly behind that in the surface regions. In turn,         phobic groups (e.g. the side chain or protecting group of an
      this observation suggests that the effective diffusion coeffi-            amino acid), this can contribute a large effect. The synthesis
      cient of the enzyme molecules inside the bead is considerably             reaction leads to transfer of these groups out of solution
      lower compared with that in aqueous solution.                             and into an environment near the solid surface, where
         It is preferable to be able to monitor the reaction con-               hydrophobic hydration is less unfavourable. After reaction,
      tinuously in real time. For this purpose, we have used an                 these groups will be less in contact with water, and this
      alternative strategy (which has also been adopted to probe                water will already be partly ordered by the nearby surface,
      reaction at different spots on a plane surface [5,6]). An amino-          or by polymer chains that form part of the support structure.
      coumarin fluorophore is first attached to the solid support               This effect is obviously greatest for the most hydrophobic
      through a carboxylic acid side chain that does not interfere              reactants, and for benzyloxycarbonyl (Z) phenylalanine can
      with fluorescence. Then, the amino group is acylated with an              be as large as 104 (change in ratio of amide to amine).
      enzyme-cleavable residue such as Ac (acetyl)-Phe. The amide                  (iii) Mutual electrostatic repulsion of charged groups on
      produced is non-fluorescent, so the bead now becomes rel-                 the solid surface will favour synthesis. In amide hydrolysis,
      atively dark. Enzymatic removal of the Ac-Phe group, for ex-              for example, ionization of the liberated amino and carboxy
      ample by chymotrypsin, liberates the free amino group again,              groups makes a major contribution in favouring the reaction
      and restores fluorescence. Hence monitoring the develop-                  in water. If the ionizable groups are attached to the solid
      ment of fluorescence gives a real-time probe of the rate of               particle, their ionization will be suppressed by mutual

      C   2006 Biochemical Society
                                                                                                                                               Biocatalysis   311

repulsion, and hence synthesis will be relatively favoured over      Figure 2 Effect of oligoglycine spacer length on rate of
hydrolysis. The effect can be seen as a change in pK of the          chymotrypsin-catalysed hydrolysis
surface-attached groups (in terms of the bulk pH), but is            From none to eight glycine residues were attached to PEGA or amino-
properly modelled as a Donnan equilibrium. The effect                propylsilane-treated CPG, followed by N-terminal Ac-Trp. The measured
is largest at low ionic strength, and is relatively small in the     rate of Ac-Trp release after addition of chymotrypsin to medium (66 µg ·
case studied with approx. 0.1 M salt.                                ml−1 ) is shown. Rates are expressed as percentage of maximum, in
                                                                     nmol · (g of support)−1 · min−1 , of 113 (CPG) and 1.56 (PEGA). (J. Deere,
Effect of spacer length attaching substrate                          A. Lalaouni, L.F. Solares, S.L. Flitsch and P.J. Halling, unpublished work).
moiety to surface
Enzyme catalysis requires binding of the substrate moiety in
the active site. Obviously, the presence of the support surface
may interfere in the process, particularly if this is only a short
distance from the site of reaction in the substrate. With this
concern in mind, some workers have attached the substrate
moiety using a ‘spacer’, a relatively flexible chain that takes
the site of reaction further away from the surface [17]. The
use of such spacers draws upon successful approaches used
in affinity chromatography and immobilized enzymes. We
have recently made a systematic study of the effects of spacer
length on chymotrypsin action (J. Deere, A. Lalaouni, L.
Solares, S.L. Flitsch and P.J. Halling, unpublished work). Two
support materials were used, PEGA and CPG (controlled                glycine residues, which may be related to the known change
pore glass). Amino groups are present in the former at the           in conformation of oligoglycines in solution.
end of the poly(ethylene glycol) chains, and were introduced
on the latter using an aminopropyl silane coupling agent.
Coupling of oligoglycine chains on to these amino groups             References
                                                                      1 Kadereit, D. and Waldmann, H. (2001) Chem. Rev. 101, 3367–3396
formed the spacers, with final attachment of an Ac-Trp
                                                                      2 Reents, R., Jeyaraj, D.A. and Waldmann, H. (2001) Adv. Synth. Catal.
residue. Chymotrypsin in solution catalysed the release of              343, 501–513
free Ac-Trp, and the rate of its appearance in the supernatant        3 Lee, Y.S. and Mrksich, M. (2002) Trends Biotechnol. 20, S14–S18
                                                                      4 Min, D.H. and Mrksich, M. (2004) Curr. Opin. Chem. Biol. 8, 554–558
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                                                                      5 Salisbury, C.M., Maly, D.J. and Ellman, J.A. (2002) J. Am. Chem. Soc. 124,
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(Figure 2). In the absence of any spacer, the rates are
                                                                      7 Berdowska, I. (2004) Clin. Chim. Acta 342, 41–69
relatively low, particularly on the PEGA resin. This probably         8 Dickinson, D.P. (2002) Crit. Rev. Oral Biol. Med. 13, 238–275
reflects the nature of the amine leaving group as well as             9 Yasuda, Y., Kaleta, J. and Bromme, D. (2005) Adv. Drug Deliv. Rev. 57,
interference from the support surface. On PEGA, there is
                                                                     10 Halling, P.J., Ulijn, R.V. and Flitsch, S.L. (2005) Curr. Opin. Biotechnol. 16,
a somewhat hindered 2-aminopropyl group, while the 1-                   385–392
aminopropyl group on CPG may be disfavoured relative                 11 Meldal, M. (2002) Biopolymers 66, 93–100
                                                                     12 Kress, J., Zanaletti, R., Amour, A., Ladlow, M., Frey, J.G. and Bradley, M.
to an amino acid residue. When an oligoglycine chain is
                                                                        (2002) Chem. Eur. J. 8, 3769–3772
introduced, two residues are sufficient for maximal rates on         13 Ulijn, R.V., Brazendale, I., Margetts, G., Flitsch, S.L., McConnell, G.,
PEGA, while at least four are required for the optimum with             Girkin, J. and Halling, P.J. (2003) J. Comb. Chem. 5, 215–217
                                                                     14 Bosma, A.Y., Ulijn, R.V., McConnell, G., Girkin, J., Halling, P.J. and Flitsch,
CPG. This probably reflects the flexibility offered by the
                                                                        S.L. (2003) Chem. Commun., 2790–2791
long poly(ethylene glycol) chains in PEGA, to the end of                                      ˜
                                                                     15 Ulijn, R.V., Baragana, B., Halling, P.J. and Flitsch, S.L. (2002) J. Am.
which the oligoglycine is attached. In contrast with CPG,               Chem. Soc. 124, 10988–10989
                                                                     16 Ulijn, R.V., Bisek, N., Halling, P.J. and Flitsch, S.L. (2003)
the amino group of the silane is close to and probably fairly
                                                                        Org. Biomol. Chem. 1, 1277–1281
fixed relative to the rigid glass surface. Hence more glycine        17 Schmitz, C. and Reetz, M.T. (1999) Org. Lett. 1, 1729–1731
residues are needed for optimal flexibility at the reaction site.
In both cases, there is a decline in rate with more than six         Received 21 November 2005

                                                                                                                              C   2006 Biochemical Society