Redesigning Trypsin: Alteration of Substrate Specificity

Craik, Charles S.; Largman, Corey; Fletcher, Thomas S.; Roczniak, Steven; Barr, Philip J.; Fletterick, Robert J.; Rutter, William J.

doi:10.1126/science.3838593

Cited by 369 publications

(199 citation statements)

References 43 publications

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“…What are the differences between the Asp-His dyad in PI-PLC and that in the classic serine proteases? Previously, the contribution of LBHB in serine proteases to catalysis was often estimated from the decrease in k cat /K m (by a factor of ϳ10 4 ) when the active site Asp was mutated to Asn (22,23). However, it is important to note that this loss cannot be attributed entirely to the loss of LBHB, for the tautomeric form of the His residue is reversed in the D102N mutant of trypsin (24).…”

Section: Dissecting the Roles Of The H-bond And The Negative Charge Omentioning

confidence: 99%

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

Zhao¹,

Liao

Tsai

2004

Journal of Biological Chemistry

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Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp 274 in the Asp 274 -His 32 dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (ϳ4 kcal/ mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp 274 . To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp 274 in the Asp 274 -His 32 dyad can be attributed to a negative charge (as in D274E and D274G-Cl ؊ ), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositolspecific phospholipase C.

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Section: Dissecting the Roles Of The H-bond And The Negative Charge Omentioning

confidence: 99%

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

Zhao¹,

Liao

Tsai

2004

Journal of Biological Chemistry

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“…Replacing the aspartate residue with asparagine in trypsin (23,24) or alanine in subtilisin (25) leads to a 10 4 -fold reduction in catalytic activity. This decrease suggests that the aspartate residue plays a critical role in catalysis.…”

mentioning

confidence: 99%

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

1998

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The sidechains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His⋯Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His⋯Asp dyad is composed of His119 and Asp121. Previously, sitedirected mutagenesis was used to show that His119 has a fundamental role-to act as an acid during catalysis of RNA cleavage , J. Am. Chem. Soc. 116,[5467][5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 Å with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, N δ rather than O δ of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3′→5′)adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2′,3′-cyclic phosphate. Replacing Asp121 decreases the values of k cat /K m and k cat for cleavage by 101-fold (D121N) and 10 2 -fold (D121A). Replacing Asp121 also decreases the values of k cat /K m and k cat for hydrolysis by 10 0.5 -fold (D121N) and 10-fold (D121A), but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His⋯Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.Keywords catalytic dyad; catalytic triad; low-barrier hydrogen bond; pH-rate profile; ribonuclease A; serine protease; site-directed mutagenesis; X-ray crystallographyThe amino acid motifs available to enzymic catalysts are diverse. Still, many enzymes fall into classes wherein great similarities exist. One well-studied class is that of the serine proteases (1). This class of enzymes has an active-site motif known as the catalytic triad, which is composed of the residues Ser⋯His⋯Asp linked by hydrogen bonds (2,3). † This work was supported by Grant GM44783 (NIH). X-ray data collection and computational facilities were supported by Grant BIR-9317398 (NSF). L.W.S. was supported by postdoctoral fellowship CA69750 (NIH). D.J.Q. was supported by Cellular and Molecular Biology Training Grant GM07215 (NIH).*Author to whom correspondence should be addressed.. ‡ Present address: School of Pharmacy, University of Wisconsin -Madison, Madison, WI 53706. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2...

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“…As suggested above, the studies with trypsin have required an additional effort to express the eukaryotic protein in a microbial host and ensure correct proteolytic processing of the zymogen in vitro. The steps en route to this goal (Craik et al, 1984) deserve some attention in their own right but cannot be discussed in detail here. Whereas subtilisin may be processed and activated autocatalytically prior to its release into the growth medium (Power et al, 1986), the trypsinogen gene product, whether secreted from animal or bacterial cells, requires activation by exogenous enteropeptidase.…”

Section: Introductionmentioning

confidence: 99%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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Redesigning Trypsin: Alteration of Substrate Specificity

Cited by 369 publications

References 43 publications

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

Protein engineering. The design, synthesis and characterization of factitious proteins

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