Almost one-third of all proteases can be classified as serine proteases, named for the nucleophilic Ser residue at the active site. This mechanistic class was originally distinguished by the presence of the AspHis-Ser “charge relay” system or “catalytic triad”.1 The Asp-His-Ser triad can be found in at least four different structural contexts, indicating that this catalytic machinery has evolved on at least four separate occasions.2 These four clans of serine proteases are typified by chymotrypsin, subtilisin, carboxypeptidase Y, and Clp protease (MEROPS nomenclature;3 Table 1). More recently, serine proteases with novel catalytic triads and dyads have been discovered, including Ser-His-Glu, Ser-Lys/His, His-Ser-His, and N-terminal Ser.2 Several of these novel serine proteases are subjects of accompanying articles in this issue. This article will review recent work on the mechanism and specificity of chymotrypsin-like enzymes, with the occasional references to pertinent experiments with subtilisin. Chymotrypsin-like proteases are the most abundant in nature, with over 240 proteases recognized in the MEROPS database.3 * E-mail: hedstrom@brandeis.edu; phone: 781-736-2333; FAX: 781-736-2349. 4501 Chem. Rev. 2002, 102, 4501−4523

Serine protease mechanism and specificity

The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of kcat and KM values of several thousand enzymes collected from the literature. We found that the “average enzyme” exhibits a kcat of ∼10 s–1 and a kcat/KM of ∼105 s–1 M–1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ∼30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit KM optimization. In accordance, subst...

The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters

Binding Energy, Specificity, and Enzymic Catalysis: The Circe Effect

What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?

Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.

Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2871%2980037-6

Determination of the individual rate constants of α-chymotrypsin-catalyzed hydrolysis with the added nucleophilic agent, 1,4-butanediol

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2872%2980299-0

The influence of the geometric properties of the active centre on the specificity of α-chymotrypsin catalysis

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2871%2980038-8

On the relationship between structure and reactivity of α‐chymotrypsin substrates

The effect of added nucleophiles (methanol and 1,4-butanediol) on the steady-state kinetics of α-chymotryptic hydrolysis of a series of N-acetyl-L-amino acid methyl esters, R-CH(NHCOCH3)C(O)OCH3, has been studied. As a result, the rate and equilibrium constants of the ‘elementary’ steps of the enzyme process have been determined.



It has also been demonstrated how the free energy–reaction coordinate profile changes if the structure (the size of the hydrocarbon chain) of the ‘chemically inert’ substrate fragment R is varied. The effects observed can be described by the following equation: 






where ΔGs and ΔGa are the free energies of formation of metastable intermediates, i.e., the enzyme–substrate complex and the acylenzyme, respectively, ΔG2≠ and ΔG3≠ are the free energies of activation for the chemical steps, i.e., enzyme acylation and acylenzyme hydrolysis, respectively; and ΔGtrans(R) is the free energy of transfer of substrate group R from water into a nonaqueous solvent.



To explain the results obtained, a mechanism for enzyme–substrate interaction is suggested according to which the potential free energy of sorption of substrate group R on the enzyme is 2 ΔGtrans(R). Such a high gain in the free energy of hydrophobic interaction may only be realized if (a) in the free enzyme the sorption region has a thermodynamically unfavorable contact with the aqueous medium, and (b) water is forced out of the active center as a result of the hydrophobic interaction of substrate group R with the enzyme. Such a model is in agreement with the published x-ray data on the structure of the crystalline enzyme.



The kinetic experiment has proved that not all the potential free energy of sorption is realized as binding force. Thus the true free energy of the binding of substrate group R with the protein does not exceed half the maximum value, both in the enzyme–substrate complex and acylenzyme.

The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

All photographic processes are based on the ability of a material to change its properties, especially transparency and color, under the action of light. These changes which arise from photochemical conversions of light sensitive compounds, result in the formation of a photographic image. There are very many light sensitive compounds; but only a few have found application in photography. Examples are 1) silver salts that are reduced to silver metal by light, 2) complex salts of iron which are converted to divalent iron by light and in turn can reduce salts of other metals, 3) bichromates of alkaline metals that can tan a gelatin carrier under the action of light, 4) diazo compounds that undergo degradation under the action of light; 5) dyes that change their color under the action of light, and 6) monomers or low molecular weight soluble polymers that under the action of light are changed into insoluble high molecular weight compounds (1–3). As things are today, light signals can be recorded and converted into visible images in any region of the spectrum.

N.F. Kazanskaya

Papers

Determination of the individual rate constants of α-chymotrypsin-catalyzed hydrolysis with the added nucleophilic agent, 1,4-butanediol

The influence of the geometric properties of the active centre on the specificity of α-chymotrypsin catalysis

On the relationship between structure and reactivity of α‐chymotrypsin substrates

The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

Application of Immobilized Enzyme Systems in Nonsilver Photography