Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.

We wish to alert the reader to errors that were inadvertantly published in our recent article. First, in the Abstract, we incorrectly state that our CD experiments included Ca (II) ion titrations of AP7-1 and AP24-1 synthetic polypeptides; these Ca (II) titrations were withheld from the final draft of the paper and are not included in the published report, but will appear elsewhere. Secondly, in our AP7-1 and AP24-1 calcite overgrowth experiments, we incorrectly reported the concentrations utilized in our study under the Materials and Methods section subheading, "Crystal Growth Analysis." The correct concentrations utilized for AP7-1, AP24-1 should read as 1.65 ϫ 10 Ϫ6

1. When (methyL2H 3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 'H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion.2. When [methyf-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. 3. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA).4. By addition of ['4C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly.5. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)-and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using ('H4)succinyl-CoA.6 . When (I -I3C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (< 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place.(1 ,4-13C2)Succinic acid and (1 ,4-'3C2)succinyl-CoA were synthesised and their I3C-NMR chemical shifts were exactly determined. 7. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonylCoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3 H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)-position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)-position into the labile a-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs.A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.

1. Samples of methylmalonyl-CoA and (2H3)methylmalonyl-CoA were prepared by a combination of chemical and enzymic methods. After ion-exchange chromatography the unlabelled methylmalonyl-CoA was pure, the deuterated substance contained 11 -12% dephospho-CoA derivative.2. The sample of unlabelled methylmalonyl-CoA was incubated in deuterated buffer with catalytic amounts of methylmalonyl-CoA mutase, epimerase, and coenzyme B1 2. The progress of the reaction was monitored directly by 'H-NMR spectroscopy at 500 MHz. After equilibrium was established,, a slow mutase-catalysed deuterium incorporation into migratable positions of succinyl-CoA was observed.3. The sample of (2H3)methylmalonyl-CoA was incubated in unlabelled buffer with a mixture of methylmalonyl-CoA mutase, epimerase and coenzyme B12. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilised samples were examined by 'H-NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl-CoA were monitored.4. At comparable methylmalonyl-CoA to succinyl-CoA conversion rates, deuterium loss from migratable positions was 4 -6 times faster than the corresponding protium loss. It is confirmed that the stereochemical error of the mutase is amplified by isotope discrimination when deuterium is in migratable positions, whereas it is diminished when protium is in migratable positions.Methylmalonyl-CoA mutase, a coenzyme-B12-dependent enzyme, catalyses the reversible interchange of the CoA thiolester group and a hydrogen atom between two adjacent carbon centers of its substrates, the coenzyme A esters of methylmalonic and succinic acids (Scheme 1). The enzyme is stereospecific for the (2R)-epimer of methylmalonyl-CoA; no reaction was discernible with the (2q-epimer within the limits of experimental error [l]. The (2R)-and (2q-epimers are rapidly interconverted by the coenzyme-B 2-independent methylmalonyl-CoA epimerase via abstraction of the a-proton (OH).