©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for a (Triosephosphate Isomerase-like) Catalytic Loop near the Active Site of Glyoxalase I (*)

Yin Lan (1), Tianfen Lu (1), Paul S. Lovett (2), Donald J. Creighton (1)(§)

From the (1) Laboratory for Chemical Dynamics, Departments of Chemistry and Biochemistry and (2) Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21228

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH (1), CH (2), CHCl (3), or CHBr (4). For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction. However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93. In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold. These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site. Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I(100-111) and Pseudomonas putida Glx I(93-105), that shows significant sequence homology to the ``catalytic loop'' of chicken muscle triosephosphate isomerase (TIM)(165-176). The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P. putida Glx I. Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity.


INTRODUCTION

The isomerase glyoxalase I (Glx I,() EC 4.4.1.5) is recognized to be a highly efficient catalyst for interconverting glutathione (GSH)-methylglyoxal thiohemiacetal and S-D-lactoylgluta-thione via a cis-enediol proton-transfer mechanism (for reviews, see Refs. 1 and 2) (Fig. R1).


Figure R1: Reaction 1.



This enzyme, together with the thioester hydrolase glyoxalase II (Glx II), plays a vital role in chemically removing cytotoxic methylglyoxal from cells as D-lactate. From a practical perspective, Glx I has been the subject of renewed interest as a possible anticancer target because of speculation that the subtle differences in the activities of the glyoxalase enzymes between normal cells and cancer cells might serve as the basis for the development of Glx I inhibitors that are tumor-selective (3, 4) , and because of the recent demonstration that GSH-based inhibitors of Glx I can be efficiently delivered into leukemia cells as the [glycyl,-glutamyl]diethyl esters (5) . Thus, obtaining detailed structural and mechanistic information on Glx I is of considerable importance, in order to provide a solid basis for inhibitor design.

Unfortunately, the recently available primary structures of human Glx I (6, 7) and Pseudomonas putida Glx I (8) do not show high levels of sequence homology with other enzymes of known structure and function. Nevertheless, whether by chance or by heredity, Glx I catalyzes a reaction that is stereochemically and mechanistically analogous to that catalyzed by triosephosphate isomerase (TIM), which interconverts dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate via a cis-enediol(ate) intermediate (9) . Interestingly, a significant fraction of the intracellular methylglyoxal that is detoxified by Glx I has been calculated to arise from the incorrect processing of the enzyme-bound enediol(ate) intermediate formed along the reaction pathway of TIM (10) . Conceivably, the two enzymes might be distantly related through evolution.

During the course of studies aimed at identifying catalytically important conformational changes in Glx I, we have uncovered evidence for a flexible peptide loop near the active site. Proteolytic susceptibility measurements and mutagenesis experiments suggest that the loop is functionally analogous to the ``catalytic loop'' of TIM. This is the first evidence that Glx I and TIM are related at the level of enzyme structure and function.


EXPERIMENTAL PROCEDURES

Materials

Pronase E (Type XIV) from Streptomyces griseus, S-D-lactoylglutathione, and phenylmethylsulfonyl fluoride were purchased from Sigma and used without further purification. The enediol analogs were synthesized as described previously (3, 4) . Glx I was purified to apparent homogeneity from human erythrocytes, following the procedure of Aronsset al.(11) . The purified enzyme was at least 95% pure, as judged by SDS-PAGE. All other reagents were of the highest purity commercially available.

Spectrophotometric Methods

Ligand-induced quenching of Glx I fluorescence was measured using an SLM 48000S spectrofluorimeter (25 °C). The fluorescence intensity of solutions containing Glx I and ligand was corrected for contributions due to buffer, and to inner filter effects arising from absorption of exciting light by ligand. The inner filter correction was obtained from the decrease in the fluorescence intensity of a standard solution of L-tryptophan in the presence of known concentrations of ligand. The dissociation constant (K ) of the ligand with the enzyme was obtained from nonlinear regression analysis of the fluorescence titration data using the following equation: F = F - R(F - F), where F is the fluorescence intensity at a given concentration of the ligand and F and F are the fluorescence intensities at zero and at saturating concentrations of ligand, respectively. The term R is the fraction of enzyme bound to ligand, defined as (0.5 (([E] + [L] + K ) - (([E] + [L] + K ) - 4[E][L])))/[E], where [L] is the total concentration of ligand and [E] is the total concentration of Glx I active sites.

Circular dichroism (CD) spectra were obtained using a Jasco J-710 spectropolarimeter. The CD spectra were corrected for contributions due to buffer and at least two scans were averaged for each spectrum.

Proteolytic Susceptibility

The cleavage of human Glx I by Pronase E was followed by SDS-PAGE. Aliquots were withdrawn from digestion mixtures as a function of time and quenched by the addition of phenylmethylsulfonyl fluoride (in 100% ethanol) to a final concentration of 9 mM. These fractions were then heat-denatured in the presence of 2-mercaptoethanol and resolved on 16.5% SDS-polyacrylamide minigels following the method of Schagger and von Jagow (12) . The molecular weight markers (Promega) spanned the range 2.5-31 kDa. The amount of protein in each lane was quantitated by the use of a Molecular Dynamics Laser Scanning Personal Densitometer. Pronase E activity was measured using casein as a substrate, following the protocol supplied by Sigma.

In order to determine the sites of proteolytic cleavage, the peptide fragments were transferred from SDS-PAGE gels to polyvinylidene difluoride membranes by electroblotting, stained with Coomassie Brilliant Blue R-250 and subjected to N-terminal analysis. Identification of the sites of proteolytic cleavage was based on analyzing at least 10 amino acids at the N terminus of the peptides.

Mutagenesis

A mutant P. putida Glx I with Asp-93 replacing Glu-93 (E93D) was prepared using plasmid pBTac1/glx I described elsewhere (8) . A 1.5-kilobase pair HindIII-EcoRI restriction fragment containing the Glx I gene was inserted into the HindIII/EcoRI site of M13mp8. Recombinant M13bacteriophage was propagated in Escherichia coli strain TG1, and single-stranded M13 DNA was purified (13) . Oligonucleotide-directed mutagenesis was performed by the method of Eckstein (14) , as described elsewhere (15) . Mutants were identified by single-base sequence analysis and confirmed by performing a full sequencing reaction. The Asp-93 Glx I mutant gene was isolated from M13mp8 on a 1.5-kilobase pair HindIII-EcoRI fragment and served as a template for polymerase chain reaction amplification to obtain a 535-base pair fragment containing the Glx I gene. The upstream oligonucleotide placed an NdeI restriction site right before the start codon of Glx I. The downstream oligonucleotide placed a BamHI site 7 nucleotides from the stop codon (TAA) of Glx I. The E93D Glx I mutant gene was isolated from the polymerase chain reaction mixture and inserted into the NdeI/BamHI site of a pET-15b vector (Novagen). Glx I was purified from E. coli BL21(DE3) transformed with this vector, using methods described elsewhere (16) .


RESULTS AND DISCUSSION

Our interest in the conformational mobility of Glx I originated with the problem of designing tight-binding inhibitors of Glx I that also serve as substrates for the hydrolase Glx II (3, 4) . Recently, we suggested that inhibitors of this type might function as potential tumor-selective anticancer agents because of the critical role that glyoxalase plays in removing toxic methylglyoxal from cells, and because of the abnormally low levels of Glx II activity in many types of cancer cells. Thus, tumor selectivity could arise from the reduced ability of cancer cells to hydrolyze the inhibitors.

In support of the feasibility of this anticancer strategy, we also reported that S-N-hydroxycarbamoyl esters of GSH (e.g. 1-4) serve as slow substrates for bovine liver Glx II and strong competitive inhibitors of human erythrocyte Glx I. Tight binding to Glx I appears to result from the fact that these compounds are stable analogs of the tightly bound enediol(ate) intermediate that forms along the reaction pathway (3, 4) , shown in Fig. D1. Moreover, the high affinity of Glx I for the enediol analogs, versus substrate/product, appears to result from differential binding of functional groups that are both near to and far from the reaction center (4) . This implies that different conformational forms of Glx I are involved in binding substrate, intermediate and product species. Indeed, Mannervik and co-workers (17) previously proposed that catalysis might be coupled to a significant change in enzyme conformation. Their hypothesis was based on the observation that binding of the competitive inhibitor S-(p-bromobenzyl)glutathione to the active site of human erythrocyte Glx I induces a 10% decrease in protein fluorescence, while binding of the product S-D-lactoylglutathione does not affect protein fluorescence.


Figure D1: Diagram 1.



Fluorescence and Circular Dichroism Measurements

In order to further evaluate this hypothesis, the enediol analog ( 1) was tested for its ability to quench the intrinsic fluorescence of human erythrocyte Glx I. Increasing concentrations of 1 progressively decrease protein fluorescence to about 89% of its original value (Fig. 1). Regression analysis of the fluorescence intensity data gave an apparent dissociation constant (K= 0.92 ± 0.32 µM) in approximate agreement with the competitive inhibition constant of 1 with Glx I (K= 1.7 ± 0.1 µM(4) ). S-D-Lactoylglutathione does not produce fluorescence quenching under saturating conditions, in accordance with the original report of Mannervik and co-workers (17) . To the extent that 1 is a reasonable analog of the enediol(ate) intermediate, the ligand-induced fluorescence quenching effects are indicative of a catalytically important change in enzyme conformation going from bound intermediate to bound product. Nevertheless, this conformational change must be subtle, since the circular dichroism spectrum of the enzyme is relatively insensitive to the presence of saturating concentrations of either 1 or of the aromatic enediol analog 4 (data not shown).


Figure 1: Fluorescence intensity of an aqueous solution of human erythrocyte Glx I (1 µM in active sites) as a function of [S-(N-methyl-N-hydroxycarbamoyl)glutathione(1)]: = 295 nm, = 346 nm. The solidline through the data is the best fit line obtained from regression analysis of the data (see ``Experimental Procedures''). Inset, fluorescence emission spectrum ( = 295 nm) of an aqueous solution of Glx I (1 µM in active sites) in presence (dottedline) and in the absence (solidline) of a near-saturating concentration of 1 (7.73 µM). Conditions: 10 mM Tris-HCl (pH 7.8), 25 °C.



Proteolytic Susceptibility Measurements

The movement of a flexible loop of contiguous amino acid residues near an active site is one type of subtle conformational change that has been proposed to accompany catalysis by such diverse enzymes as phosphoglycerate mutase (18), lactate dehydrogenase (19) , and TIM (20) . This prompted a search for an analogous peptide loop near the active site of Glx I by determining the susceptibility of the enzyme to proteolytic cleavage in the presence and in the absence of enediol analogs. For example, the catalytically important peptide loop near the active site of TIM is known to be susceptible to proteolytic cleavage by subtilisin (21) .

Consistent with the presence of a flexible peptide loop near the active site of human Glx I, the presence of either 2 or 3 diminishes the rate of proteolytic cleavage by Pronase E (e.g.Fig. 2 ). In the absence of enediol analog, Pronase E cleaves Glx I into two major peptide fragments that migrate on SDS-PAGE gels at 9.5 and 5.1 kDa. Near-saturating concentrations of 3 decrease the first order rate constant for loss of Glx I from a value of 3.0 10 min to a value of 9.3 10 min (Fig. 3). Since Pronase E is not inhibited by 3 at the concentration used in the proteolytic digestion studies, the protective effect of 3 must be due to binding to Glx I. In contrast, near-saturating concentrations of the product S-D-lactoylglutathione actually increases the rate of loss of intact enzyme (k = 4.5 10 min) ( Fig. 2and Fig. 3). Edman analysis of the 9.5-kDa species showed that it was actually composed of two peptides resulting from cleavage of the enzyme at the C-side of Ala-92 and Leu-93 (Fig. 4). Analysis of the 5.1-kDa fragment showed that it arose from cleavage of the enzyme at the C-side of Ala-23, near to the N terminus of the untreated enzyme (Ser-18).() No evidence could be found for proteolytic cleavage of P. putida Glx I by Pronase E. This appears to reflect some critical difference in the primary structures of the human and bacterial enzymes at the sites of proteolytic cleavage of the human enzyme (Fig. 4).


Figure 2: SDS-PAGE gels showing the time course of proteolytic digestion of human erythrocyte Glx I (443 µg/ml) by Pronase E (44 µg/ml, 2.2 10 units) in solutions containing no added ligand (panel A), containing a near-saturating concentration of S-D-lactoylglutathione (17.3 mM) (panel B) and containing a near-saturating concentration of S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (3) (19.3 µM) (panel C). Conditions: 30 mM Tris-HCl buffer (pH 7.5), 37 °C.




Figure 3: First-order rate plots for the loss of intact human erythrocyte Glx I (443 µg/ml), due to the action of Pronase E (44 µg/ml, 2.2 10 units), in solutions containing no added ligand (cir-cles), containing a near-saturating concentration of S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (3) (19.3 µM) (squares), and containing a near-saturating concentration of S-D-lactoylglutathione (17.3 mM) (triangles). Conditions: 30 mM Tris-HCl buffer (pH 7.5), 37 °C.




Figure 4: Amino acid sequences deduced from the published gene structures of human colon/human leukocyte Glx I (6, 7) (upper) and P. putida Glx I (8) (lower). a, N terminus of purified human erythrocyte Glx I deduced from Edman analysis. b, N terminus of the 5.1-kDa peptide fragment generated from Pronase E digestion of human erythrocyte Glx I. c, N termini of the two 9.5-kDa peptide fragments generated from Pronase E digestion of human erythrocyte Glx I. d, mutation of Glu-93 to Asp-93 in P. putida Glx I dramatically reduces catalytic activity. The overlined region shows significant sequence similarity to the catalytic loop of TIM.



In native proteins, proteolytic cleavage sites are normally observed at or near loops or turns characterized by high flexibility. This was clearly demonstrated by Fontana and co-workers (22) when they established an exact correspondence between the sites of proteolytic cleavage in thermolysin and regions of high mobility in the enzyme protein, indicated by the poor resolution of these regions in the x-ray structure of the crystalline enzyme. Thus, Glx I probably has at least one such flexible loop containing Leu-93 and Ser-94 (Fig. 4). With respect to the minimum size of the loop, proteases generally bind at primary and secondary sites of peptide segments containing 6-8 amino acid residues (23) . The partial protection from proteolysis afforded by the enediol analogs can be explained by closure of the loop over bound enediol analog. The fact that S-D-lactoylglutathione stimulates proteolytic cleavage implies that this ligand binds to and stabilizes a loop-open form of Glx I that is more accessible to binding by Pronase E. These observations are consistent with a model of catalysis in which a ``catalytic'' loop near the active site functions to stabilize the bound enediol(ate) intermediate, and conversion to S-D-lactoylglutathione is coupled to opening of the loop in order to facilitate product release. An analogous role has been proposed for the peptide loop near the active site of TIM (20) .

Mutagenesis

Indeed, TIM and Glx I may be distantly related.() The sites of proteolytic cleavage at Ala-92/Leu-93 in human Glx I are near a region(100-111) showing significant sequence homology to the catalytic loop of TIM (Fig. 5).


Figure 5: Comparison of the catalytic loop region of TIM with an analogous region in Glx I.



This region is one of several highly conserved regions between human and P. putida Glx I, implying an important role in the structure/function of the enzyme (Fig. 4). The comparison with the TIM sequence also suggests that Glu-100 of human Glx I and Glu-93 of P. putida Glx I might contribute the active site base involved in the enediol-proton-transfer mechanism proposed for Glx I (Fig. R1). These residues correspond in position to Glu-165 of TIM. The carboxyl group of Glu-165 undoubtedly catalyzes the proton-transfer reaction associated with isomerization, on the basis of affinity labeling (25, 26, 27) and site-directed mutagenesis studies (28) .

In order to test this hypothesis, site-directed mutagenesis was used to construct a mutant form of P. putida Glx I in which Glu-93 is replaced by Asp, giving E93D Glx I. We reasoned that if Glu-93 contributes the active site base the mutant enzyme would show little catalytic activity, based on the fact that the specific activity of E165D TIM is about 10-fold lower than that of wild-type TIM (28) . Indeed, E93D Glx I is catalytically inactive within the limits of detection: specific activity 0.02% of wild-type enzyme. The mutant enzyme appears to be fully folded in solution, as the CD spectrum is similar to that of wild-type Glx I. These observations are consistent with a catalytic role for Glu-93 in the mechanism of action of P. putida Glx I.

Conclusions

Glx I appears to contain a flexible peptide loop near the active site that is structurally and functionally analogous to the catalytic loop of TIM. If correct, this would be evidence for the long sought-after linkage between the glyoxalase and glycolytic pathways, recently discussed by Vander Jagt (2) .


FOOTNOTES

*
This work was supported by American Cancer Society Grant BE83 (to D. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-455-2518; Fax: 410-455-2608.

The abbreviations used are: Glx, glyoxalase; TIM, triosephosphate isomerase; PAGE, polyacrylamide gel electrophoresis.

It is not clear why the N terminus of the untreated enzyme does not correspond to that predicted from the published gene structure. The disparity might be due to partial proteolysis during purification, as the molecular mass of the enzyme obtained immediately from the affinity column (24.5 kDa) was observed to decrease to a mass of 19.5 kDa after overnight dialysis of the column effluent. Nevertheless, the specific activities of the 24.5- and 19.5-kDa species were found to be similar: 1,640 and 1,360 units/mg, respectively. These values are also close to the literature value (1,000 units/mg), for which the molecular mass of the human enzyme was reported to be 26 kDa (SDS-PAGE) (11).

Chicken muscle TIM and human Glx I show about 17% sequence identity over 185 amino acid residues, at the borderline between common ancestry and chance similarity (24).


ACKNOWLEDGEMENTS

We thank Kevin Lawton of the University of Maryland Blood Bank for supplying the outdated human erythrocytes used in the preparation of Glx I, and Ray Sowder of the Frederick Cancer Center for N-terminal analyses.


REFERENCES
  1. Creighton, D. J., and Pourmotabbed, T.(1988) in Molecular Structure and Energetics: Mechanistic Principles of Enzyme Activity (Liebman, J. K., and Greenberg, A., eds) Vol. 9, pp. 353-386, VCH Publishers, New York
  2. Vander Jagt, D. L.(1989) in Coenzymes and Cofactors: Glutathione (Dolphin, D., Poulson, R., and Avramovic, O., eds) Vol. 3, pp. 597-641, John Wiley & Sons, New York
  3. Hamilton, D. S., and Creighton, D. J.(1992) J. Biol. Chem. 267, 24933-24936 [Abstract/Free Full Text]
  4. Murthy, N. S. R. K., Bakeris, T., Kavarana, M. J., Hamilton, D. S., Lan, Y., and Creighton, D. J.(1994) J. Med. Chem. 37, 2161-2166 [Medline] [Order article via Infotrieve]
  5. Lo, T. W. C., and Thornalley, P. J.(1992) Biochem. Pharmacol. 44, 2357-2363 [CrossRef][Medline] [Order article via Infotrieve]
  6. Kim, N.-S., Umezawa, Y., Ohmura, S., and Kato, S.(1993) J. Biol. Chem. 268, 11217-11221 [Abstract/Free Full Text]
  7. Ranganathan, S., Walsh, E. S., Godwin, A. K., and Tew, K. D.(1993) J. Biol. Chem. 268, 5661-5667 [Abstract/Free Full Text]
  8. Lu, T., Creighton, D. J., Antoine, M., Fenselau, C., and Lovett, P. S. (1994) Gene (Amst.) 150, 93-96 [CrossRef][Medline] [Order article via Infotrieve]
  9. Rose, I. A., and O'Connell, E. L.(1961) J. Biol. Chem. 236, 3086-3092 [Medline] [Order article via Infotrieve]
  10. Richard, J. P.(1991) Biochemistry 30, 4581-4585 [Medline] [Order article via Infotrieve]
  11. Aronss, A.-C., Tibbelin, G., and Mannervik, G.(1979) Anal. Biochem. 92, 390-393 [Medline] [Order article via Infotrieve]
  12. Schagger, H., and von Jagow, G.(1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  13. Maniatis, T., Fritsch, E. F., and Sambrook, J.(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. Taylor, J. W., Ott, J., and Eckstein, F.(1985) Nucleic Acids Res. 13, 8765-8785 [Abstract]
  15. Duvall, E. J., Ambulos, N. P., and Lovett, P. S.(1987) J. Bacteriol. 169, 4235-4241 [Medline] [Order article via Infotrieve]
  16. Rhee, H.-I., Murata, K., and Kimura, A.(1987) Biochem. Biophys. Res. Commun. 147, 831-838 [Medline] [Order article via Infotrieve]
  17. Sellin, S., Eriksson, L. E. G., and Mannervik, B.(1982) Biochemistry 21, 4850-4857 [Medline] [Order article via Infotrieve]
  18. Winn, S. I., Watson, H. C., Harkin, R. N., and Fothergill, L. A.(1981) Philos. Trans. R. Soc. Lond. B 293, 121-130 [Medline] [Order article via Infotrieve]
  19. Clarke, A. R., Wigley, D. B., Chia, W. N., Barstow, D., Atkinson, T., and Holbrook, J. J.(1986) Nature 324, 699-702 [Medline] [Order article via Infotrieve]
  20. Pompliano, D. L., Peyman, A., and Knowles, J. R.(1990) Biochemistry 29, 3186-3194 [Medline] [Order article via Infotrieve]
  21. Sun, A.-Q., Yüksel, K. U., and Gracy, R. W.(1993) J. Biol. Chem. 268, 26872-26878 [Abstract/Free Full Text]
  22. Fontana, A., Fassina, G., Vita, C., Dalzoppo, D., Zamai, M., and Zambonin, M.(1986) Biochemistry 25, 1847-1851 [Medline] [Order article via Infotrieve]
  23. Ottesen, M.(1967) Annu. Rev. Biochem. 36, 55-76
  24. Doolittle, R. F.(1985) Sci. Am. 253, 88-99 [Medline] [Order article via Infotrieve]
  25. Miller, J. C., and Waley, S. G.(1971) Biochem. J. 123, 163-170 [Medline] [Order article via Infotrieve]
  26. Hartman, F. C.(1971) Biochemistry 10, 146-154 [Medline] [Order article via Infotrieve]
  27. De la Mare, S., Coulson, A. F. W., Knowles, J. R., Priddle, J. D., and Offord, R. E.(1972) Biochem. J. 129, 321-331 [Medline] [Order article via Infotrieve]
  28. Raines, R. T., Sutton, E. L., Straus, D. R., Gilbert, W., and Knowles, J. R.(1986) Biochemistry 25, 7142-7154 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.